Hemiasterlin derivatives for conjugation and therapy

ABSTRACT

Provided herein are hemiasterlin derivatives, conjugates thereof, compositions comprising the derivatives or conjugates thereof, methods of producing the derivatives and conjugates thereof, and methods of using the derivatives, conjugates, and compositions for the treatment of cell proliferation. The derivatives, conjugates, and compositions are useful in methods of treatment and prevention of cell proliferation and cancer, methods of detection of cell proliferation and cancer, and methods of diagnosis of cell proliferation and cancer. In an embodiment, the hemiasterlin derivatives are according to Formula 1000: 
                         
or a pharmaceutically acceptable salt, solvate, or tautomer thereof, wherein Ar, L, W 1 , W 4 , W 5 , SG, and R are as described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/011,388, filed on Jan. 29, 2016, which claims priority to, andthe benefit of, U.S. Provisional Application No. 62/110,390, filed onJan. 30, 2015, which are incorporated herein by reference in theirentirety.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

This application includes an electronically submitted sequence listingin .txt format. The .txt file is named “108843.00305_ST25.txt” createdon Apr. 14, 2023 and is 617 bytes in size. The sequence listingcontained in this .txt file is part of the specification and is herebyincorporated by reference herein in its entirety.

FIELD

Provided herein are hemiasterlin derivatives, conjugates thereof,compositions comprising the derivatives or conjugates thereof, methodsof producing the derivatives and conjugates thereof, and methods ofusing the derivatives, conjugates, and compositions for the treatment ofcell proliferation. The derivatives, conjugates, and compositions areuseful in methods of treatment and prevention of cell proliferation andcancer, methods of detection of cell proliferation and cancer, andmethods of diagnosis of cell proliferation and cancer.

BACKGROUND

Hemiasterlins are a class of tripeptides modified from the originalnatural product hemiasterlin. Hemiasterlin is isolated from marinesponges Cymbastela sp., Hemiasterella minor, Siphonochalina sp., andAuletta sp. (Talpir et al., Tetrahedron Letters, vol. 35, no. 25, pp.4453-4456, 1994).

Hemiasterlins are pseudopeptides which are inhibitors of tubulinpolymerization, sharing an antimitotic mechanism of action withdolastatins and cryptophycins. Noncompetitive binding at the vinblastinesite on tubulin has been demonstrated. Hemiasterlins are in general poorpermeability glycoprotein (pGP) substrates, rendering them effectiveagainst tumors that overexpress pGP as a resistance mechanism. (Loganzoet al., Cancer Research, vol 63, pp. 1838-1845, 15 Apr. 2003).

Extensive modification of natural hemiasterlin demonstrated key featurescontributing to the nanomolar activity of this class against a widevariety of tumor cell lines. Two derivatives, E7974, an N-terminalpiperidine derivative developed at Eisai, and HTI-286, an N-terminalphenyl developed at Wyeth, entered Phase I trials. Encouraging resultswere presented in 2007 for E7974. (Madajewicz et al., “A phase I trialof E7974 administered on days 1 and 15 of a 28-day cycle in patientswith solid malignancies,” presented at American Society of ClinicalOncology Annual Meeting; Jun. 1-5, 2007; Chicago, Ill.; and Zojwalla etal., “A phase I trial of E7974 administered on days 1, 8, and 15 of a28-day cycle in patients with solid malignancies,” presented at AmericanSociety of Clinical Oncology Annual Meeting; Jun. 1-5, 2007; Chicago,Ill.: both summarized in Rocha-Lima et al., Cancer, Sep. 1, 2012 pp.4262-4270). However, no results have been reported for HTI-286 to date.

In addition, conjugation of HTI-286 at the C-terminus to a gastrindecapeptide VLALAEEEAYGWNleDF-NH₂ for tumor targeting is described inTarsova et al., United States patent application publication number US2005/0171014 A1. Reported activity, however, was very weak.

SUMMARY

Provided herein are hemiasterlin derivatives, conjugates thereof,compositions comprising the derivatives or conjugates thereof, methodsof producing the derivatives and conjugates thereof, and methods ofusing the derivatives, conjugates, and compositions for the treatment ofcell proliferation. The derivatives, conjugates, and compositions areuseful in methods of treatment and prevention of cell proliferation andcancer, methods of detection of cell proliferation and cancer, andmethods of diagnosis of cell proliferation and cancer.

In one aspect, provided herein is a compound according to Formula 1000:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   X is

-   -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is absent or an eliminator group;    -   each RT is a release trigger group, in the backbone of Formula        1000 or bonded to EG, wherein each RT is optional;    -   RT¹ is a release trigger group, or a cleavable linker, or RT¹ is        absent;    -   HP is a single bond, absent, or a divalent hydrophilic group;    -   HP¹ is a single bond, absent, a divalent hydrophilic group, or

-   -    where R^(HP) is a monovalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is hydrogen, a terminal conjugating group, or a divalent        residue of a terminal conjugating group;    -   or, in the alternative, W¹, W², W³, W⁴, W⁵, EG, RT, HP, SG, and        R combine to form —H.

In one aspect, provided herein is a conjugate comprising a compounddescribed herein (e.g., a compound according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b)linked to a second compound.

In an aspect, provided herein is a pharmaceutical compositioncomprising:

-   -   a compound (e.g., a compound according to any of Formulas        1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or        1-8b) or conjugate (e.g., a conjugate according to any of        Formulas C1-C17b, E1, F1-F17b, and G1-G17b) as described herein;        and    -   a pharmaceutically acceptable excipient, carrier, or diluent.

In an aspect, provided herein is a method of inhibiting tubulinpolymerization in a subject in need thereof comprising administering aneffective amount of a compound (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b), conjugate (e.g., a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b), or composition comprising a compoundor conjugate, as described herein, to the subject.

In an aspect, provided herein is a method of treating cell proliferationor cancer in a subject in need thereof comprising administering aneffective amount of a compound (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b), conjugate (e.g., a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b), or composition comprising a compoundor conjugate, as described herein, to the subject.

In an aspect, provided herein is a method of producing a conjugate,comprising contacting a compound described herein (e.g., a compoundaccording to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, andI-XIXb-2 or 101-111b) with a second compound under conditions suitablefor conjugating the second compound with the compound described herein(e.g., a compound according to any of Formulas 1000-1000b, 1001-1001b,1002-1002b, and I-XIXb-2 or 101-111b); wherein the second compoundcomprises a modified amino acid comprising an alkyne, strained alkene,tetrazine, thiol, maleimide, carbonyl, oxyamine, or azide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides results of a cell killing assay described in detailherein. Racemic [R/S,S,S] Compound 1 is evaluated as a conjugate on thetrastuzumab heavy chain at F404 and also on the light chain at S7. Allother conjugates in FIG. 1 are on the trastuzumab heavy chain at F404.The relative cell viability of SKBR3 cells is plotted againstconcentration for trastuzumab (exes), trastuzumab F404 [S,S,S] Compound1 conjugate (filled squares), trastuzumab F404 racemic [R/S,S,S]Compound 1 conjugate (split squares), trastuzumab S7 racemic [R/S,S,S]Compound 1 conjugate (split circles), and a trastuzumab auristatin(MMAF) conjugate (open triangles).

FIG. 2 a provides results of a cell killing assay described in detailherein. In FIG. 2 a relative cell viability is plotted againstconcentration of [S,S,S] Compound 1 (filled squares) and [R,S,S]Compound 1 (open squares) for SKBR3 cells in panel (a), MDA-MB-453 cellsin panel (b), and MDA-MB-468 cells in panel (c).

FIG. 2 b provides results of a cell killing assay described in detailherein. In FIG. 2 b relative cell viability is plotted againstconcentration of [S,S,S] Compound 1 (filled squares) and [R,S,S]Compound 1 (open squares) for HTC116 cells in panel (a), HT29 cells inpanel (b), and SKCO1 cells in panel (c).

FIG. 2 c provides results of a cell killing assay described in detailherein. In FIG. 2 c relative cell viability is plotted againstconcentration of [S,S,S] Compound 1 (filled squares) and [R,S,S]Compound 1 (open squares) for MDA-MB-435 cells in panel (a), SUDHL6cells in panel (b), and OMP2 cells in panel (c).

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are compounds (e.g., according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b),conjugates thereof (e.g., according to any of Formulas C1-C17b, E1,F1-F17b, and G1-G17b), compositions comprising the compounds orconjugates thereof, methods of producing the compounds and conjugatesthereof, and methods of using the compounds, conjugates, andcompositions. The compounds, conjugates, and compositions are useful inmethods of treatment and prevention of cell proliferation and cancer,methods of detection of cell proliferation and cancer, and methods ofdiagnosis of cell proliferation and cancer.

Definitions

When referring to the compounds provided herein, the following termshave the following meanings unless indicated otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of ordinary skill in the art.In the event that there is a plurality of definitions for a term herein,those in this section prevail unless stated otherwise. Unless specifiedotherwise, where a term is defined as being unsubstituted orsubstituted, the groups in the list of substituents are unsubstituted.For example, an alkyl group can be substituted with a cycloalkyl groupand the cycloalkyl group is not further substituted.

The term “alkyl,” as used herein, unless otherwise specified, refers toa saturated straight or branched hydrocarbon which can be substitutedwith halo groups. In certain embodiments, the alkyl group is a primary,secondary, or tertiary hydrocarbon. In certain embodiments, the alkylgroup includes one to ten carbon atoms, i.e., C₁ to C₁₀ alkyl. Incertain embodiments, the alkyl group is, for example, methyl, CF₃, CCl₃,CFCl₂, CF₂Cl, ethyl, CH₂CF₃, CF₂CF₃, propyl, isopropyl, butyl, isobutyl,secbutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl,3-methylpentyl, 2,2-dimethylbutyl, or 2,3-dimethylbutyl. The termincludes both substituted and unsubstituted alkyl groups, includinghalogenated alkyl groups. In certain embodiments, the alkyl group is afluorinated alkyl group. In certain embodiments, the alkyl group can besubstituted with at least one (in another example with 1, 2, 3, 4, or 5)halogen (fluoro, chloro, bromo or iodo), oxo, epoxy, hydroxyl,alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, cycloalkyl, aralkyl,sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, sulfonate, phosphonic acid, phosphate, or phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl,” as used herein, and unless otherwise specified,refers to a saturated straight or branched hydrocarbon having one to sixcarbon atoms, i.e., C₁ to C₆ alkyl. In certain embodiments, the loweralkyl group is a primary, secondary, or tertiary hydrocarbon. The termincludes both substituted and unsubstituted moieties.

The term “upper alkyl,” as used herein, and unless otherwise specified,refers to a saturated straight or branched hydrocarbon having seven tothirty carbon atoms, i.e., C₇ to C₃₀ alkyl. In certain embodiments, theupper alkyl group is a primary, secondary, or tertiary hydrocarbon. Theterm includes both substituted and unsubstituted moieties.

The term “alkylcarbonyl” refers to the group —C(O)(alkyl) where alkyl isas defined herein.

The term “alkylsulfanyl” refers to the group —S(alkyl) where alkyl is asdefined herein.

The term “carboxylene” refers to a —C(O)O— or —OC(O)— group.

The term “cycloalkylsulfanyl” refers to the group —S(cycloalkyl) wherecycloalkyl is as defined herein.

The term “arylsulfanyl” refers to the group —S(aryl) where aryl is asdefined herein.

The term “alkylsulfonyl” refers to the group —S(O)₂(alkyl) where alkylis as defined herein.

The term “cycloalkylsulfonyl” refers to the group —S(O)₂(cycloalkyl)where cycloalkyl is as defined herein.

The term “arylsulfonyl” refers to the group —S(O)₂(aryl) where aryl isas defined herein.

The term “cycloalkyl,” as used herein, unless otherwise specified,refers to a saturated monocyclic or polycyclic hydrocarbon. In certainembodiments, cycloalkyl includes fused, bridged, and spiro ring systems.In certain embodiments, the cycloalkyl group includes three to tencarbon atoms, i.e., C₃ to C₁₀ cycloalkyl. In some embodiments, thecycloalkyl has from 3 to 15 (C₃₋₁₅), from 3 to 10 (C₃₋₁₀), or from 3 to7 (C₃₋₇) carbon atoms. In certain embodiments, the cycloalkyl group is,for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl,bicyclo[2.2.1]heptyl, decalinyl or adamantyl. The term includes bothsubstituted and unsubstituted cycloalkyl groups, including halogenatedcycloalkyl groups. In certain embodiments, the cycloalkyl group is afluorinated cycloalkyl group. In certain embodiments, the cycloalkylgroup can be substituted with at least one (in another example with 1,2, 3, 4, or 5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy,hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g alkylamino, dialkylamino, arylamino,etc.), alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

The term “cycloalkylalkyl” refers to an alkyl group as defined hereinsubstituted with at least one (in some embodiments, one or two)cycloalkyl groups as defined herein.

The term “cycloalkylcarbonyl” refers to the group —C(O)(cycloalkyl)where cycloalkyl is as defined herein.

“Alkylene” refers to divalent saturated aliphatic hydrocarbon groups,including those having from one to eleven carbon atoms which can bestraight-chained or branched. In certain embodiments, the alkylene groupcontains 1 to 10 carbon atoms. The term includes both substituted andunsubstituted moieties. In certain embodiments, alkylene is, forexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers(e.g., —CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like. The term includeshalogenated alkylene groups. In certain embodiments, the alkylene groupis a fluorinated alkylene group. In certain embodiments, the alkylenegroup can be substituted with at least one (in another example with 1,2, 3, 4, or 5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy,hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g. alkylamino, dialkylamino, arylamino,etc.), alkylaryl, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,sulfonate, phosphonic acid, phosphate, and phosphonate, eitherunprotected, or protected as necessary.

“Alkenyl” refers to monovalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms,including from 2 to 8 carbon atoms, or from 2 to 6 carbon atoms, whichcan be straight-chained or branched and having at least 1, includingfrom 1 to 2, site of olefinic unsaturation. The term includes bothsubstituted and unsubstituted moieties. In certain embodiments, alkenylis, for example, ethenyl (i.e., vinyl, or —CH═CH₂), n-propenyl(—CH₂CH═CH₂), isopropenyl (—C(CH₃)═CH₂), and the like. The term includeshalogenated alkenyl groups. In certain embodiments, the alkenyl group isa fluorinated alkenyl group. In certain embodiments, the alkenyl groupcan be substituted with at least one (in another example with 1, 2, 3,4, or 5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy, hydroxyl,alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g. alkylamino, dialkylamino, arylamino,etc.), alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

The term “cycloalkenyl,” as used herein, unless otherwise specified,refers to an unsaturated (but not aromatic) cyclic hydrocarbon. Incertain embodiments, cycloalkenyl refers to mono- or multicyclic ringsystems that include at least one double bond. In certain embodiments,cycloalkyl includes fused, bridged, and spiro ring systems. In certainembodiments, the cycloalkyl group includes at least three carbon atoms,including three to ten carbon atoms, i.e., C₃ to C₁₀ cycloalkyl. In someembodiments, the cycloalkenyl has from 3 to 10 (C₃₋₁₀), or from 4 to 7(C₄₋₇) carbon atoms. The term includes both substituted andunsubstituted cycloalkenyl groups, including halogenated cycloalkenylgroups. In certain embodiments, the cycloalkenyl group is a fluorinatedcycloalkenyl group. In certain embodiments, the cycloalkenyl group canbe substituted with at least one (in another example with 1, 2, 3, 4, or5) halogen (fluoro, chloro, bromo or iodo), oxo, epoxy, hydroxyl,alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g. alkylamino, dialkylamino, arylamino,etc.), alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,phosphonic acid, phosphate, or phosphonate, either unprotected, orprotected as necessary.

“Alkenylene” refers to divalent olefinically unsaturated hydrocarbongroups, in certain embodiments, having up to about 11 carbon atoms orfrom 2 to 6 carbon atoms which can be straight-chained or branched andhaving at least 1 or from 1 to 2 sites of olefinic unsaturation. Incertain embodiments, alkenylene is, for example, ethenylene (—CH═CH—),the propenylene isomers (e.g., —CH═CHCH₂— and —C(CH₃)═CH— and—CH═C(CH₃)—) and the like. The term includes both substituted andunsubstituted alkenylene groups, including halogenated alkenylenegroups. In certain embodiments, the alkenylene group is a fluorinatedalkenylene group. Non-limiting examples of moieties with which thealkenylene group can be substituted with at least one (in anotherexample with 1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or iodo),oxo, epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, sulfonate, phosphonic acid, phosphate, or phosphonate,either unprotected, or protected as necessary.

“Alkynyl” refers to acetylenically unsaturated hydrocarbon groups, incertain embodiments, having up to about 11 carbon atoms or from 2 to 6carbon atoms which can be straight-chained or branched and having atleast 1 or from 1 to 2 sites of alkynyl unsaturation. In certainembodiments, alkynyl is, for example, acetylenic, ethynyl (—C≡CH),propargyl (—CH₂C≡CH), and the like. The term includes both substitutedand unsubstituted alkynyl groups, including halogenated alkynyl groups.In certain embodiments, the alkynyl group is a fluorinated alkynylgroup. In certain embodiments, the alkynyl group can be substituted withat least one (in another example with 1, 2, 3, 4, or 5) halogen (fluoro,chloro, bromo or iodo), oxo, epoxy, hydroxyl, alkylcarbonyl,cycloalkylcarbonyl, arylcarbonyl, sulfanyl, alkylsulfanyl,cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl, cycloalkylsulfonyl,arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido, amino (as definedherein, e.g. alkylamino, dialkylamino, arylamino, etc.), alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate, phosphonicacid, phosphate, or phosphonate, either unprotected, or protected asnecessary.

The term “aryl,” as used herein, and unless otherwise specified, refersto a monovalent six- to fourteen-membered, mono-, bi-, ortri-carbocyclic ring, wherein the monocyclic ring is aromatic and atleast one of the rings in the bicyclic and tricyclic ring is aromatic.The aryl group can be bonded to the rest of the molecule through anycarbon in the ring system. In an embodiment, an aryl group is a C₆-C₁₂aryl group. In an embodiment, an aryl group is phenyl, indanyl, ornaphthyl. The term includes both substituted and unsubstituted moieties.In certain embodiments, an aryl group can be substituted with one ormore (for example 1, 2, 3, 4, or 5) moieties independently selected fromthe group halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl,hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g. alkylamino, dialkylamino, arylamino,etc.), alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,phosphonic acid, phosphate, and phosphonate, either unprotected, orprotected as necessary, as known to those skilled in the art, forexample, as taught in Greene, et al., Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

The term “arylcarbonyl” refers to the group —C(O)(aryl) where aryl is asdefined herein.

The term “aryloxy” refers to the group —OR′ where R′ is aryl, as definedherein.

The term “aryloxyalkyl” refers to an alkyl group as defined hereinsubstituted with at least one (in some embodiments one or two) aryloxygroups as defined herein.

“Alkoxy” and “alkoxyl” refer to the group —OR′ where R′ is alkyl orcycloalkyl as defined herein. In certain embodiments, alkoxy and alkoxylgroups include, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, cyclopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy,n-hexoxy, 1,2-dimethylbutoxy, and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with atleast one (in another embodiment, one or two) alkoxy groups as definedherein.

“Alkoxycarbonyl” refers to a radical —C(O)-alkoxy where alkoxy is asdefined herein.

“Alkoxycarbonylalkyl” refers to an alkyl group substituted with at leastone, in another example 1 or 2, alkoxycarbonyl groups, as definedherein.

“Amino” refers to the group —NR^(1′)R^(2′) or —NR^(1′)—, wherein R^(1′)and R^(2′) are independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocyclic, aryl, or heteroaryl, each ofwhich is as defined herein. In an embodiment, “Amino” is —NH₂ or —NH—.

“Carboxyl” or “carboxy” refers to the radical —C(O)OH.

The term “alkylamino” or “arylamino” refers to an amino group that hasone alkyl or aryl substituent, respectively, e.g. —NHCH₃, and—NH(phenyl). In certain embodiments, the alkyl substituent is loweralkyl. In another embodiment, the alkyl or lower alkyl is unsubstituted.

The term “dialkylamino” refers to an amino group that has two alkylsubstituents, e.g. —N(CH₃)₂. In certain embodiments, the alkylsubstituent is lower alkyl. In another embodiment, the alkyl or loweralkyl is unsubstituted.

The term “diarylamino” refers to an amino group that has two arylsubstituents.

“Halogen” or “halo” refers to chloro, bromo, fluoro, or iodo.

“Thioalkoxy” refers to the group —SR′ where R′ is alkyl or cycloalkyl.

The term “heterocyclo” or “heterocyclic” refers to a monovalentmonocyclic non-aromatic ring system and/or multicyclic ring system thatcontains at least one non-aromatic ring, wherein one or more of thenon-aromatic ring atoms are heteroatoms independently selected from O,S, or N and the remaining ring atoms are carbon atoms and where themulticyclic ring system further comprises a carbocyclic or heterocyclic,aromatic or nonaromatic ring. In certain embodiments, the heterocyclo orheterocyclic group has from 3 to 20, from 3 to 15, from 3 to 10, from 3to 8, from 4 to 7, or from 5 to 6 ring atoms. Heterocyclo groups arebonded to the rest of the molecule through a non-aromatic ring. Incertain embodiments, the heterocyclo is a monocyclic, bicyclic,tricyclic, or tetracyclic ring system, which may include a fused,spirocyclic, or bridged ring system, and in which the nitrogen or sulfuratoms may be optionally oxidized, the nitrogen atoms may be optionallyquaternized, and some rings may be partially or fully saturated, oraromatic. The heterocyclo may be attached to the main structure at anyheteroatom or carbon atom of the non-aromatic ring which results in thecreation of a stable compound. In certain embodiments, heterocyclic isazepinyl, benzodioxanyl, benzodioxolyl, benzofuranonyl, benzopyranonyl,benzopyranyl, benzotetrahydrofuranyl, benzotetrahydrothienyl,benzothiopyranyl, benzoxazinyl, O-carbolinyl, chromanyl, chromonyl,cinnolinyl, coumarinyl, decahydroisoquinolinyl, dihydrobenzisothiazinyl,dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl,dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl,furanonyl, imidazolidinyl, imidazolinyl, indolinyl,isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isochromanyl,isocoumarinyl, isoindolinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl,oxazolidinyl, oxiranyl, piperazinyl, piperidinyl, 4-piperidonyl,pyrazolidinyl, pyrazolinyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl,tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydrothienyl, thiamorpholinyl, thiazolidinyl, tetrahydroquinolinyl,and 1,3,5-trithianyl. In certain embodiments, heterocyclic may also beoptionally substituted as described herein. In certain embodiments, theheterocyclic group can be substituted with at least one (in anotherexample with 1, 2, 3, 4, or 5) halogen (fluoro, chloro, bromo or iodo),oxo, epoxy, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl,alkoxycarbonyl, alkoxycarbonylalkyl, sulfanyl, alkylsulfanyl,cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl, cycloalkylsulfonyl,arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido, amino (as definedherein, e.g., alkylamino, dialkylamino, arylamino, etc.), alkoxy,aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate, phosphonicacid, phosphate, or phosphonate, either unprotected, or protected asnecessary.

The term “heteroaryl” refers to a monovalent monocyclic aromatic groupand/or multicyclic group that contains at least one aromatic ring,wherein the monocyclic ring contains one or more heteroatomsindependently selected from O, S and N in the ring and where themulticyclic ring system comprises at least one aromatic ring and furthercomprises a carbocyclic or heterocyclic, aromatic or nonaromatic ringand where one or more of the ring atoms in the multicyclic ring systemis a heteroatom independently selected from O, S and N. Heteroarylgroups are bonded to the rest of the molecule through an aromatic ring.Each ring of a heteroaryl group can contain one or two O atoms, one ortwo S atoms, and/or one to four N atoms, provided that the total numberof heteroatoms in each ring is four or less and each ring contains atleast one carbon atom. In certain embodiments, the heteroaryl has from 5to 20, from 5 to 15, or from 5 to 10 ring atoms. In certain embodiments,monocyclic heteroaryl groups include, but are not limited to, furanyl,imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl,oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl,pyrrolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, triazinyl andtriazolyl. In certain embodiments, bicyclic heteroaryl groups include,but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl,benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl,imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl,isobenzothienyl, isoindolyl, isoquinolinyl, isothiazolyl,naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl,pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl,thiadiazolopyrimidyl, and thienopyridyl. In certain embodiments,tricyclic heteroaryl groups include, but are not limited to, acridinyl,benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl,phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyland xanthenyl. In certain embodiments, the heteroaryl group can besubstituted with at least one (in another example with 1, 2, 3, 4, or 5)group halogen (fluoro, chloro, bromo or iodo), alkyl, haloalkyl,hydroxyl, alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, sulfanyl,alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl, alkylsulfonyl,cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl, carbamoyl, sulfonamido,amino (as defined herein, e.g. alkylamino, dialkylamino, arylamino,etc.), alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, sulfonate,phosphonic acid, phosphate, and phosphonate, either unprotected, orprotected as necessary.

The term “alkylaryl” refers to an aryl group with an alkyl substituent,wherein aryl and alkyl are as defined herein. The term “aralkyl” or“arylalkyl” refers to an alkyl group with an aryl substituent, whereinaryl and alkyl are as defined herein.

The term “phenylene,” as used herein, and unless otherwise specified,refers to a divalent phenyl group and includes both substituted andunsubstituted moieties. In certain embodiments, phenylene group can besubstituted with one or more (for example 1, 2, 3, 4, or 5) moietiesindependently selected from the group halogen (fluoro, chloro, bromo oriodo), alkyl, haloalkyl, hydroxyl, alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, sulfanyl, alkylsulfanyl, cycloalkylsulfanyl, arylsulfanyl,alkylsulfonyl, cycloalkylsulfonyl, arylsulfonyl, aminocarbonyl,carbamoyl, sulfonamido, amino (as defined herein, e.g. alkylamino,dialkylamino, arylamino, etc.), alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, sulfonate, phosphonic acid, phosphate, and phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991. Whenphenylene is used in the context of an EG group, the phenylene issubstituted with 1, 2, 3, or 4 R^(EG) groups, as defined herein, and/orwith 1 or 2 —O—[RT] groups, and/or with 1 or 2 —CH₂OC(O)[RT] groups,where RT is as defined herein.

The term “protecting group” as used herein and unless otherwise definedrefers to a group that is added to an oxygen, nitrogen or phosphorusatom to prevent its further reaction or for other purposes. A widevariety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis.

“Pharmaceutically acceptable salt” refers to any salt of a compoundprovided herein which retains its biological properties and which is nottoxic or otherwise undesirable for pharmaceutical use. Such salts may bederived from a variety of organic and inorganic counter-ions well knownin the art. Such salts include, but are not limited to: (1) acidaddition salts formed with organic or inorganic acids such ashydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic,acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic,cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic,succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric,benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic,phthalic, lauric, methanesulfonic, ethanesulfonic,1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic,4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic,camphoric, camphorsulfonic,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic,3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric,gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic,cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) baseaddition salts formed when an acidic proton present in the parentcompound either (a) is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion or an aluminum ion, or alkali metal oralkaline earth metal hydroxides, such as sodium, potassium, calcium,magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b)coordinates with an organic base, such as aliphatic, alicyclic, oraromatic organic amines, such as ammonia, methylamine, dimethylamine,diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine,ethylenediamine, lysine, arginine, ornithine, choline,N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine,N-benzylphenethylamine, N-methylglucamine piperazine,tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and thelike.

Pharmaceutically acceptable salts further include, by way of exampleonly and without limitation, sodium, potassium, calcium, magnesium,ammonium, tetraalkylammonium and the like, and when the compoundcontains a basic functionality, salts of non-toxic organic or inorganicacids, such as hydrohalides, e.g. hydrochloride and hydrobromide,sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate,trichloroacetate, propionate, hexanoate, cyclopentylpropionate,glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate,ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate,3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate,laurate, methanesulfonate (mesylate), ethanesulfonate,1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate(besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate,4-toluenesulfonate, camphorate, camphorsulfonate,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate,3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate,gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate,cyclohexylsulfamate, quinate, muconate and the like.

The term “acyl” refers to a group of the formula —C(O)R′, wherein R′ isalkyl (including lower alkyl); cycloalkyl; cycloalkylalkyl;cycloalkenyl; aryl; arylalkyl (including benzyl); substituted alkyl(including lower alkyl and for example alkoxyalkyl and aryloxyalkyl);heterocyclo; heterocycloalkyl; heteroaryl; and heteroarylalkyl; wherethe cycloalkyl, cycloalkenyl, aryl, heterocyclo, and heteroaryl may besubstituted. In certain embodiments, aryl groups in the acyl or esterscomprise a phenyl group. In certain embodiments, acyl groups include,for example, acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl,propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoroheptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxybenzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenylcarbonyl, isovaleryl,1-pyrrolidinecarbonyl, and 4-phenylbenzoyl.

The term “amino acid” refers to naturally occurring and synthetic α, β,γ, or δ amino acids, and includes but is not limited to, amino acidsfound in proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In certain embodiments, the amino acid is in theL-configuration. In certain embodiments, the amino acid is in theD-configuration. In certain embodiments, the amino acid is provided as asubstituent of a compound described herein, wherein the amino acid is aresidue selected from alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl,phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl,glutaroyl, lysinyl, argininyl, histidinyl, β-alanyl, β-valinyl,β-leucinyl, β-isoleuccinyl, β-prolinyl, β-phenylalaninyl,β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl,β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl,β-glutaroyl, β-lysinyl, β-argininyl, or β-histidinyl.

The term “amino acid derivative” refers to a group derivable from anaturally or non-naturally occurring amino acid, as described andexemplified herein. Amino acid derivatives are apparent to those ofskill in the art and include, but are not limited to, ester, aminoalcohol, amino aldehyde, amino lactone, and N-methyl derivatives ofnaturally and non-naturally occurring amino acids. In an embodiment, acompound described herein comprises an amino acid derivative, whereinthe amino acid derivative is —NR^(X)-G(S_(C))—C(O)-Q¹-, wherein Q¹ is—S—, —NR^(Y)—, or —O—, R^(Y) is hydrogen or alkyl, S_(C) is a side chainof a naturally occurring or non-naturally occurring amino acid, G isC₁-C₂ alkylene, and R^(X) is hydrogen or R^(X) and S_(C), together withthe atoms to which they are attached, combine to form a five-memberedheterocyclic ring. In an embodiment, an amino acid derivative isprovided as a substituent of a compound described herein, wherein thesubstituent is —O—C(O)-G(S_(C))—NH-Q²-, wherein Q² is a single bond or—O—, S_(C) is a side chain of a naturally occurring or non-naturallyoccurring amino acid and G is C₁-C₂ alkylene. In certain embodiments, Q²and Sc, together with the atoms to which they are attached, combine toform a five-membered heterocyclic ring. In certain embodiments, G is C₁alkylene and S_(C) is hydrogen, alkyl, arylalkyl, heterocycloalkyl,carboxylalkyl, heteroarylalkyl, aminoalkyl, hydroxylalkyl,aminoiminoaminoalkyl, aminocarbonylalkyl, sulfanylalkyl, carbamoylalkyl,alkylsulfanylalkyl, or hydroxylarylalkyl. In an embodiment, an aminoacid derivative is provided as a substituent of a compound describedherein, wherein the amino acid derivative is in the D-configuration. Inan embodiment, an amino acid derivative is provided as a substituent ofa compound described herein, wherein the amino acid derivative is in theL-configuration.

The term “alkylheterocyclo” refers to a heterocyclo group with an alkylsubstituent. The term “heterocycloalkyl” refers to an alkyl group with aheterocyclo substituent.

As used herein, the term “carboxylalkyl” refers to an alkyl substitutedwith at least 1, in another example 1 or 2, carboxy, where alkyl is asdescribed herein.

The term “alkylheteroaryl” refers to a heteroaryl group with an alkylsubstituent. The term “heteroarylalkyl” refers to an alkyl group with aheteroaryl substituent.

As used herein, the term “aminoalkyl” refers to an alkyl groupsubstituted with at least 1, in another example 1 or 2, aminosubstituent(s), where alkyl and amino are as described herein.

As used herein, the terms “hydroxylalkyl” and “hydroxyalkyl” refer to analkyl group substituted with at least 1, in another example 1 or 2,hydroxyl, where alkyl is as described herein.

As used herein, the term “aminoiminoaminoalkyl” refers to an alkylsubstituted with at least 1, in another example 1 or 2,-amino-C(NH)-amino, where alkyl and amino are as described herein.

The term aminocarbonyl refers to the group —C(O)(amino) where amino isas defined herein.

As used herein, the term “aminocarbonylalkyl” refers to an alkylsubstituted with at least 1, in another example 1 or 2, —C(O)-amino,where alkyl and amino are as described herein.

As used herein, the term “sulfanylalkyl” refers to an alkyl substitutedwith at least 1, in another example 1 or 2, —SH, where alkyl is asdescribed herein.

The term “carbamoyl” refers to a —NRC(OR′, where R is hydrogen or alkyland R′ is alkyl, cycloalkyl, heterocyclo, heteroaryl, or aryl, asdefined herein.

As used herein, the term “carbamoylalkyl” refers to an alkyl substitutedwith at least 1, in another example 1 or 2, carbamoyl groups, as definedherein.

As used herein, the term “alkylsulfanylalkyl” refers to an alkylsubstituted with at least 1, in another example 1 or 2, —S-alkyl, wherealkyl is as described herein.

As used herein, the term “hydroxylarylalkyl” refers to the group-alkyl-aryl-OH, where alkyl and aryl are as described herein.

The term “sulfonic acid” refers to the group —S(O)₂OH.

The term “sulfate” refers to the group —OS(O)₂OR where R is alkyl orarylalkyl.

The term “sulfonate” refers to the group —S(O)₂OR where R is alkyl orarylalkyl.

The term “sulfonamido” refers to the group —S(O)₂NRR′ where R ishydrogen or alkyl and R′ is alkyl, cycloalkyl, heterocyclo, heteroaryl,or aryl, as defined herein.

The term “phosphate” refers to the group —OP(O)(OR)₂ where each R isindependently alkyl or arylalkyl.

The term “phosphonic acid” refers to —P(O)(OH)₂.

The term “phosphonate” refers to the group —P(O)(OR)₂ where each R isindependently alkyl or arylalkyl.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is amodified amino acid. Additionally, such “polypeptides,” “peptides” and“proteins” include amino acid chains of any length, including fulllength proteins, wherein the amino acid residues are linked by covalentpeptide bonds.

Many of the compounds and conjugates described herein have chiralcenters. The present disclosure encompasses each stereoisomer of eachcompound or conjugate with each possible stereochemistry at each chiralcenter. Certain compounds are identified by stereochemical notation thatis known to those of skill. In particular embodiments, stereochemistryis identified with R and S notation for each chiral center, from left toright as depicted in formula 1000, 1001, 1002, and (I), etc. or formula(C1), F1, and G1, etc. For instance, the notation [R,S,S] indicates R, Sand S stereochemistry at the chiral centers of formula (I) from left toright, beginning with the methylamino substituent position and endingwith the isopropyl substituent position. Similarly, the notation [S,S,S]indicates S, S and S stereochemistry at the chiral centers of formula(I) from left to right. Further, the notation racemic [R/S,S,S]indicates a mixture of [R,S,S] and [S,S,S] compounds. For othercompounds and conjugates herein, the notation can be applied tocorresponding structures.

The term “substantially free of” or “substantially in the absence of,”when used in connection with an article (including, but not limited to,a compound, a salt thereof, a solvate thereof, a solid form thereof, andthe like), refers to the article that includes at least 85% or 90% byweight, in certain embodiments, 95%, 98%, 99%, or 100% by weight, of thedesignated article. For example, the term “substantially free of” or“substantially in the absence of” with respect to a composition canrefer to a composition that includes at least 85% or 90% by weight, incertain embodiments, 95%, 98%, 99%, or 100% by weight, of a designatedstereoisomer of a compound. In certain embodiments, in the methods andcompounds provided herein, the compounds are substantially free ofundesignated stereoisomers or other compounds. For another example, theterm “substantially free of” or “substantially in the absence of” withrespect to a solid form can refer to a solid form that includes at least85% or 90% by weight, in certain embodiments, 95%, 98%, 99%, or 100% byweight, of the designated solid form. In certain embodiments, in themethods and compounds provided herein, the solid form is substantiallyfree of other solid forms.

Similarly, the term “isolated” with respect to a composition refers to acomposition that includes at least 85%, 90%, 95%, 98%, or 99% to 100% byweight, of a designated compound, the remainder comprising otherchemical species or stereoisomers. Similarly, the term “isolated” withrespect to a solid form of a compound refers to a solid that includes atleast 85%, 90%, 95%, 98%, or 99% to 100% by weight, of such solid formof the compound, the remainder comprising other solid forms of thecompound, other compounds, solvents, and/or other impurities.

“Solvate” refers to a compound provided herein or a salt thereof, thatfurther includes a stoichiometric or non-stoichiometric amount ofsolvent bound by non-covalent intermolecular forces. Where the solventis water, the solvate is a hydrate.

“Isotopic composition” refers to the amount of each isotope present fora given atom, and “natural isotopic composition” refers to the naturallyoccurring isotopic composition or abundance for a given atom. Atomscontaining their natural isotopic composition may also be referred toherein as “non-enriched” atoms. Unless otherwise designated, the atomsof the compounds recited herein are meant to represent any stableisotope of that atom. For example, unless otherwise stated, when aposition is designated specifically as “H” or “hydrogen,” the positionis understood to have hydrogen at its natural isotopic composition.

“Isotopic enrichment” refers to the percentage of incorporation of anamount of a specific isotope at a given atom in a molecule in the placeof that atom's natural isotopic abundance. For example, deuteriumenrichment of 1% at a given position means that 1% of the molecules in agiven sample contain deuterium at the specified position. Because thenaturally occurring distribution of deuterium is about 0.0156%,deuterium enrichment at any position in a compound synthesized usingnon-enriched starting materials is about 0.0156%. The isotopicenrichment of the compounds provided herein can be determined usingconventional analytical methods known to one of ordinary skill in theart, including mass spectrometry and nuclear magnetic resonancespectroscopy.

“Isotopically enriched” refers to an atom having an isotopic compositionother than the natural isotopic composition of that atom. “Isotopicallyenriched” may also refer to a compound containing at least one atomhaving an isotopic composition other than the natural isotopiccomposition of that atom.

As used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “amino,” “carboxyl,”“alkylamino,” “arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise deuterium at one or more positions where hydrogen atoms arepresent, and wherein the deuterium composition of the atom or atoms isother than the natural isotopic composition.

Also as used herein, “alkyl,” “cycloalkyl,” “alkenyl,” “cycloalkenyl,”“alkynyl,” “aryl,” “alkoxy,” “alkoxycarbonyl,” “carboxyl,” “alkylamino,”“arylamino,” “thioalkyoxy,” “heterocyclyl,” “heteroaryl,”“alkylheterocyclyl,” “alkylheteroaryl,” “acyl,” “aralkyl,” “alkaryl,”“purine,” “pyrimidine,” “carboxyl” and “amino acid” groups optionallycomprise carbon-13 at an amount other than the natural isotopiccomposition.

As used herein, EC₅₀ refers to a dosage, concentration or amount of aparticular test compound that elicits a dose-dependent response at 50%of maximal expression of a particular response that is induced, provokedor potentiated by the particular test compound.

As used herein, the IC₅₀ refers to an amount, concentration or dosage ofa particular test compound that achieves a 50% inhibition of a maximalresponse in an assay that measures such response.

“Cancer” refers to cellular-proliferative disease states, including butnot limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiatedsmall cell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hanlartoma, inesothelioma; Gastrointestinal: colon (coloncarcinoma, colon adenocarcinoma, colorectal adenocarcinoma), esophagus(squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma),stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors,Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma,Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra(squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma),prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma,embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma,interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors,lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pineal oma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], platinum-resistant ovarian, granulosa-thecal cell tumors,Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma, ovarianadenocarcinoma), vulva (squamous cell carcinoma, intraepithelialcarcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma (embryonalrhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood(myeloid leukemia [acute and chronic], acute lymphoblastic leukemia,chronic lymphocytic leukemia, myeloproliferative diseases, multiplemyeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin'slymphoma [malignant lymphoma]; Skin: melanoma, malignant melanoma, basalcell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;Adrenal Glands: neuroblastoma; a lymphoma; large cell lymphoma; diffusemixed histiocytic and lymphocytic lymphoma; follicular B cell lymphoma;and breast (breast cancer which overexpresses Her2, triple-negativebreast cancer). Thus, the term “cancerous cell” as provided herein,includes a cell afflicted by any one of the above-identified conditions.As used herein, the terms “subject” and “patient” are usedinterchangeably herein. The terms “subject” and “subjects” refer to ananimal, such as a mammal including a non-primate (e.g., a cow, pig,horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, achimpanzee, and a human), and for example, a human. In certainembodiments, the subject is refractory or non-responsive to currenttreatments for cell proliferation and/or cancer. In another embodiment,the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or apet (e.g., a dog or a cat). In certain embodiments, the subject is ahuman.

As used herein, the terms “therapeutic agent” and “therapeutic agents”refer to any agent(s) which can be used in the treatment or preventionof a disease or condition, or one or more symptoms thereof. In certainembodiments, the term “therapeutic agent” includes a compound providedherein. In certain embodiments, a therapeutic agent is an agent which isknown to be useful for, or has been or is currently being used for thetreatment or prevention of a or condition, or one or more symptomsthereof.

“Therapeutically effective amount” refers to an amount of a compound orcomposition that, when administered to a subject for treating a diseaseor condition, is sufficient to effect such treatment for the disease orcondition. A “therapeutically effective amount” can vary depending on,inter alia, the compound, the disease or condition and its severity, andthe age, weight, etc., of the subject to be treated.

“Treating” or “treatment” of any disease or condition refers, in certainembodiments, to ameliorating a disease or condition that exists in asubject. In another embodiment, “treating” or “treatment” includesameliorating at least one physical parameter, which may be indiscernibleby the subject. In yet another embodiment, “treating” or “treatment”includes modulating the disease or condition, either physically (e.g.,stabilization of a discernible symptom) or physiologically (e.g.,stabilization of a physical parameter) or both. In yet anotherembodiment, “treating” or “treatment” includes delaying the onset of thedisease or condition.

As used herein, the terms “prophylactic agent” and “prophylactic agents”as used refer to any agent(s) which can be used in the prevention of adisease or condition, or one or more symptoms thereof. In certainembodiments, the term “prophylactic agent” includes a compound providedherein. In certain other embodiments, the term “prophylactic agent” doesnot refer a compound provided herein. For example, a prophylactic agentis an agent which is known to be useful for, or has been or is currentlybeing used to prevent or impede the onset, development, progressionand/or severity of a disease or condition.

As used herein, the phrase “prophylactically effective amount” refers tothe amount of a therapy (e.g., prophylactic agent) which is sufficientto result in the prevention or reduction of the development, recurrenceor onset of one or more symptoms associated with a disease or condition,or to enhance or improve the prophylactic effect(s) of another therapy(e.g., another prophylactic agent).

The term “antibody” refers to any macromolecule that would be recognizedas an antibody by those of skill in the art. Antibodies share commonproperties including binding and at least one polypeptide chain that issubstantially identical to a polypeptide chain that can be encoded byany of the immunoglobulin genes recognized by those of skill in the art.The immunoglobulin genes include, but are not limited to, the κ, λ, α, γ(IgG1, IgG2, IgG3, and IgG4), δ, ε and μ constant region genes, as wellas the immunoglobulin variable region genes. The term includesfull-length antibodies and antibody fragments recognized by those ofskill in the art, and variants thereof.

The term “antibody fragment” refers to any form of an antibody otherthan the full-length form. Antibody fragments herein include antibodiesthat are smaller components that exist within full-length antibodies,and antibodies that have been engineered. Antibody fragments include butare not limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like (Maynard & Georgiou, 2000, Annu.Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.9:395-402).

The term “immunoglobulin (Ig)” refers to a protein consisting of one ormore polypeptides substantially encoded by one of the immunoglobulingenes, or a protein substantially identical thereto in amino acidsequence. Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains including but not limited to V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

The term “immunoglobulin (Ig) domain” refers to a protein domainconsisting of a polypeptide substantially encoded by an immunoglobulingene. Ig domains include but are not limited to V_(H), Cγ1, Cγ2, Cγ3,V_(L), and C_(L).

The term “variable region” of an antibody refers to a polypeptide orpolypeptides composed of the V_(H) immunoglobulin domain, the V_(L)immunoglobulin domains, or the V_(H) and V_(L) immunoglobulin domains.Variable region may refer to this or these polypeptides in isolation, asan Fv fragment, as a scFv fragment, as this region in the context of alarger antibody fragment, or as this region in the context of afull-length antibody or an alternative, non-antibody scaffold molecule.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called Complementarity DeterminingRegions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three or four CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The constant domains are not typically involved directly in binding anantibody to an antigen, but exhibit various effector functions.Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies or immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement.

The term “conjugate” refers to any compound that can be formed byconjugating a compound described herein to a second compound. The secondcompound can be a small molecule or a macromolecule. In someembodiments, the second compound is a bioactive molecule including, butnot limited to, a protein, a peptide, a nucleic active or a hybridthereof. In some embodiments, the second compound is a polymer such aspolyethylene glycol. In some embodiments, the second compound is atherapeutic agent, including a commercially available drug. In someembodiments, the second compound is a label that can recognize and bindto specific targets, such as a molecular payload that is harmful totarget cells or a label useful for detection or diagnosis. In someembodiments, the compound described herein is connected to the secondcompound via a linker. In some embodiments, the compound describedherein is directly connected to the second compound without a linker. Inanother embodiment the second compound is a small molecule; amacromolecule; bioactive molecule including, but not limited to, aprotein, a peptide, a nucleic active or a hybrid thereof; a polymer suchas polyethylene glycol; a therapeutic agent, including a commerciallyavailable drug; or a label that can recognize and bind to specifictargets, such as a molecular payload that is harmful to target cells ora label useful for detection or diagnosis. In another embodiment, thesecond compound comprises a modified amino acid comprising an alkyne,strained alkene, tetrazine, thiol, maleimide, carbonyl, oxyamine, orazide.

The term “variant protein sequence” refers to a protein sequence thathas one or more residues that differ in amino acid identity from anothersimilar protein sequence. Said similar protein sequence may be thenatural wild type protein sequence, or another variant of the wild typesequence. Variants include proteins that have one or more amino acidinsertions, deletions or substitutions. Variants also include proteinsthat have one or more post-translationally modified amino acids.

The term “parent antibody” refers to an antibody known to those of skillin the art that is modified according to the description providedherein. The modification can be physical, i.e., chemically orbiochemically replacing or modifying one or more amino acids of theparent antibody to yield an antibody within the scope of the presentdescription. The modification can also be conceptual, i.e., using thesequence of one or more polypeptide chains of the parent antibody todesign an antibody comprising one or more site-specific modified aminoacids according to the present description. Parent antibodies can benaturally occurring antibodies or antibodies designed or developed in alaboratory. Parent antibodies can also be artificial or engineeredantibodies, e.g., chimeric or humanized antibodies.

The term “conservatively modified variant” refers to a protein thatdiffers from a related protein by conservative substitutions in aminoacid sequence. One of skill will recognize that individualsubstitutions, deletions or additions to a peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M).        See, e.g., Creighton, Proteins: Structures and Molecular        Properties, W H Freeman & Co.; 2nd edition (December 1993).

The terms “identical” or “identity,” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same. Sequences are “substantially identical” if they havea percentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, optionally about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, or about 95% identity over a specifiedregion), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. The identity can exist over a region that is at leastabout 50 amino acids or nucleotides in length, or over a region that is75-100 amino acids or nucleotides in length, or, where not specified,across the entire sequence or a polypeptide. In the case of antibodies,identity can be measured outside the variable CDRs. Optimal alignment ofsequences for comparison can be conducted, including but not limited to,by the local homology algorithm of Smith and Waterman (1970) Adv. Appl.Math. 2:482c, by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff. In some embodiments, the BLAST algorithm is typically performedwith the “low complexity” filter turned on.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, in another embodimentless than about 0.01, and in another embodiment less than about 0.001.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acids such as proline, aminoacid analogs and amino acid mimetics that function in a manner similarto naturally occurring amino acids.

Naturally encoded amino acids are the proteinogenic amino acids known tothose of skill in the art. They include the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and the less common pyrrolysine andselenocysteine. Naturally encoded amino acids include post-translationalvariants of the 22 naturally occurring amino acids such as prenylatedamino acids, isoprenylated amino acids, myrisoylated amino acids,palmitoylated amino acids, N-linked glycosylated amino acids, O-linkedglycosylated amino acids, phosphorylated amino acids and acylated aminoacids.

The term “modified amino acid” refers to an amino acid that is not aproteinogenic amino acid, or a post-translationally modified variantthereof. In particular, the term refers to an amino acid that is not oneof the 20 common amino acids or pyrrolysine or selenocysteine, orpost-translationally modified variants thereof.

The term “strained alkene” refers to a molecule comprising an alkenemoiety that is capable of reacting with tetrazine in a tetrazineligation. Exemplary tetrazine ligations are described in Blackman etal., 2008, J. Am. Chem. Soc. 130:13518-13519. Examples includetrans-cyclooctenes and norbornenes. Useful compounds include, but arenot limited to, trans-cyclooctene, (E)-cyclooct-4-enol,(E)-cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate,5-norbornene-2-acetic acid succinimidyl ester, and5-norbomene-2-endo-acetic acid.

The term “tetrazine” refers to a compound or group comprising thefollowing structure:

wherein R²⁰¹ is lower alkyl. For example, R²⁰¹ can be methyl, ethyl, orpropyl. In certain aspects, R²⁰¹ is methyl.

Compounds

In certain embodiments, the compound is not of formula (101), (101a), or(101b) and the conjugate does not comprise the compound of formula(101), (101a), or (101b). In certain embodiments, the compound is not offormula (101a), and the conjugate does not comprise the compound offormula (101a). In certain embodiments where X is

or the compound is not the compound of formula (101), (101a), or (101b)and the conjugate does not comprise the compound of formula (101),(101a), or (101b). In certain embodiments where X is

the compound is not the compound of formula (101a), and the conjugatedoes not comprise the compound of formula (101a). In certainembodiments, the compound is not of formula (101), (101a), or (101b). Incertain embodiments, the compound is not of formula (101a). In certainembodiments where X is

the compound is not the compound of formula (101), (101a), or (101b). Incertain embodiments where X is

the compound is not the compound of formula (101a). In certainembodiments, the conjugate does not comprise the compound of formula(101), (101a), or (101b). In certain embodiments, the conjugate does notcomprise the compound of formula (101a). In certain embodiments where Xis

the conjugate does not comprise the compound of formula (101), (101a),or (101b). In certain embodiments where X is

the conjugate does not comprise the compound of formula (101a).

When a range of formula are used, for example I-XIXb-2, each formulawithin that range is included and is as if it were explicitly listed,including where the roman numeral is followed by, for example, “a,”“-1,” etc. For example, I-XIXb-2 includes Va, XIV, and XIXa-1, etc.

In an embodiment, provided herein is a compound according to Formula I:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is an eliminator group;    -   each RT is a release trigger group, in the backbone of        Formula (I) or bonded to EG, wherein one RT is optional;    -   HP is a single bond, absent, or a divalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is hydrogen, a terminal conjugating group, or a divalent        residue of a terminal conjugating group;    -   or, in the alternative, W¹, W², W³, W⁴, W⁵, EG, RT, HP, SG, and        R combine to form —H.

In one embodiment, provided herein is a compound of Formula 1000according to 1001:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is absent or an eliminator group;    -   RT¹ is a release trigger group or a cleavable linker;    -   RT is a release trigger group bonded to EG; and wherein RT is        optional;    -   HP¹ is single bond, absent, a divalent hydrophilic group, or

-   -    where R^(SG) is a monovalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is hydrogen, a terminal conjugating group, or a divalent        residue of a terminal conjugating group.

In one embodiment, provided herein is a compound of Formula 1000according to 1002:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is absent or an eliminator group;    -   RT¹ is a release trigger group or a cleavable linker;    -   RT is a release trigger group bonded to EG; and wherein RT is        optional;    -   HP¹ is single bond, absent, a divalent hydrophilic group, or

-   -    where R^(SG) is a monovalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is hydrogen, a terminal conjugating group, or a divalent        residue of a terminal conjugating group.

In certain embodiments, a conjugating group can be used to conjugate amodified Hemiasterlin as described herein (e.g., according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b) to any molecular entity capable of reacting with the conjugatinggroup to form the conjugate. In certain embodiments, the conjugatinggroup is designated R herein. The conjugating group can be directly orindirectly linked to the modified Hemiasterlin as described herein(e.g., according to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b,and I-XIXb-2, 101-111b, or 1-8b) via one or more attaching groups,eliminator groups, release trigger groups, hydrophobic groups, and/orspacer groups.

Attaching Groups

Attaching groups facilitate incorporation of eliminator groups, releasetrigger groups, hydrophobic groups, spacer groups, and/or conjugatinggroups into a compound, such as a modified Hemiasterlin as describedherein (e.g., according to any of Formulas 1000-1000b, 1001-1001b,1002-1002b, and I-XIXb-2, 101-111b, or 1-8b). Useful attaching groupsare known to, and are apparent to, those of skill in the art. Examplesof useful attaching groups are provided herein. In certain embodiments,attaching groups are designated W¹, W², W³, W⁴, or W⁵. In certainembodiments, an attaching group can comprise a divalent ester, divalentether, divalent amide, divalent amine, alkylene, arylene, sulfide,disulfide, —C(O)—, or a combination thereof. In certain embodiments anattaching group can comprise —C(O)—, —O—, —C(O)O—, —OC(O)—, —C(O)NH—,—C(O)NH-alkyl-, —OC(O)NH—, —SC(O)NH—, —NH—, —N(alkyl)-,—N(R)-alkylene-N(R)— (where each R is independently H or alkyl),—N(CH₃)CH₂CH₂N(CH₃)—, —CH₂—, —CH₂CH₂—, —CH(CH₃)—, —C(CH₃)₂—,—CH₂CH₂CH₂—, phenylene, —NHCH₂CH₂C(O)—, —C(O)CH₂CH₂NH—, —S—, —S—S—,—OCH₂CH₂O—, or the reverse (e.g. —NHC(O)—) thereof, or a combinationthereof.

Eliminator Groups

Eliminator groups facilitate separation of a biologically active portionof a compound or conjugate described herein from the remainder of thecompound or conjugate in vivo and/or in vitro. Eliminator groups canalso facilitate separation of a biologically active portion of acompound or conjugate described herein in conjunction with a releasetrigger group. For example, the eliminator group and the release triggergroup can react in a Releasing Reaction to release a biologically activeportion of a compound or conjugate described herein from the compound orconjugate in vivo and/or in vitro. Upon initiation of the releasingreaction by the release trigger, the eliminator group cleaves thebiologically active moiety, or a prodrug form of the biologically activemoiety, and forms a stable, non-toxic entity that has no further effecton the activity of the biologically active moiety.

In certain embodiments, the eliminator group is designated EG herein.Useful eliminator groups include those described herein. In certainembodiments, the eliminator group comprises a phenylene, a —C(O)—, anamino, or a combination thereof. In certain embodiments, the eliminatorgroup is:

wherein each R^(EG) is independently selected from the group consistingof hydrogen, alkyl, biphenyl, —CF₃, —NO₂, —CN, fluoro, bromo, chloro,alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylamino-C(O)—. In the second and third structures, those of skillwill recognize that EG is bonded to an RT that is not within thebackbone of the e.g. formula 1000, or (I), as indicated in the abovedescription of formula 1000 and (I). In some embodiments, each R^(EG) isindependently selected from the group consisting of hydrogen, alkyl,biphenyl, —CF₃, alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—,alkylamino-C(O)— and dialkylamino-C(O)—. In further embodiments, eachR^(EG) is independently selected from the group consisting of hydrogen,—NO₂, —CN, fluoro, bromo, and chloro.

Release Trigger Groups and Cleavable Linkers

In certain embodiments, release trigger groups facilitate separation ofa biologically active portion of a compound or conjugate describedherein from the remainder of the compound or conjugate in vivo and/or invitro. In certain embodiments, release trigger groups can alsofacilitate separation of a biologically active portion of a compound orconjugate described herein in conjunction with an eliminator group. Insome embodiments, the eliminator group and the release trigger group canreact in a Releasing Reaction to release a biologically active portionof a compound or conjugate described herein from the compound orconjugate in vivo and/or in vitro. In certain embodiments, the releasetrigger can act through a biologically-driven reaction with hightumor:nontumor specificity, such as the proteolytic action of an enzymeoverexpressed in a tumor environment.

In certain embodiments, the release trigger group is designated RTherein. In certain embodiments, RT is divalent and bonded within thebackbone of formula (I) or 1000. In other embodiments, RT is monovalentand bonded to EG as depicted above. Useful release trigger groupsinclude those described herein. In certain embodiments, the releasetrigger group comprises a residue of a natural or non-natural amino acidor residue of a sugar ring. In certain embodiments, the release triggergroup comprises a residue of a natural or non-natural amino acid orresidue of a sugar ring.

In some embodiments, the release trigger group is derived from a linkerprecursor selected from the group consisting of dipeptides, tripeptides,tetrapeptides, and pentapeptides, each of which comprises onecitrulline. Exemplary dipeptides include, but are not limited to,valine-citrulline (vc or val-cit), and N-methyl-valine-citrulline(Me-val-cit). Exemplary tripeptides include, but are not limited to,glycine-valine-citrulline (gly-val-cit). In some embodiments, therelease trigger group is derived from a linker precursor selected fromthe group consisting of valine-citrulline, N-methyl-valine-citrulline,and glycine-valine-citrulline, In certain embodiments, the releasetrigger group is:

Those of skill will recognize that the first structure is divalent andcan be bonded within the backbone of formula (I) or 1000, and that thesecond structure is monovalent and can be bonded to EG as depicted informula (I) and 1000 above.

Cleavable linkers facilitate separation of a biologically active portionof a compound or conjugate described herein from the remainder of thecompound or conjugate in vivo and/or in vitro. In certain embodiments,the release trigger can act through a biologically-driven reaction withhigh tumor:nontumor specificity, such as the proteolytic action of anenzyme overexpressed in a tumor environment. In certain embodiments, thecleavable linker is designated RT¹ herein. Useful cleavable linkersinclude those described herein. In some embodiments, the cleavablelinker is derived from a linker precursor selected from the groupconsisting of dipeptides, tripeptides, tetrapeptides, and pentapeptides.In such embodiments, the linker can be cleaved by a protease. Exemplarydipeptides include, but are not limited to, valine-alanine (VA orVal-Ala); valine-glutamic acid (Val-Glu); alanine-phenylalanine (AF orAla-Phe); phenylalanine-lysine (FK or Phe-Lys); andphenylalanine-homolysine (Phe-homoLys). Exemplary tripeptides include,but are not limited to glycine-glycine-glycine (Gly-Gly-Gly). In certainembodiments, the cleavable linker is derived from a linker precursorselected from the group consisting of dipeptides and tripeptides. Incertain embodiments, the cleavable linker is derived from a dipeptide.In certain embodiments, the cleavable linker is derived from atripeptide. In certain embodiments the cleavable linker is derived froma linker precursor derived from valine-alanine, valine-glutamic acid,phenylalanine-homolysine, phenylalanine-lysine,phenylalanine-homolysine, or glycine-glycine-glycine.

In certain embodiments the cleavable linker is derived from a linkerprecursor selected from the group consisting of dipeptides, tripeptides,tetrapeptides, and pentapeptides; or is

where aa is a natural or non-natural amino acid residue; or is

where the

ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon atoms. Incertain embodiments the cleavable linker is derived from a linkerprecursor selected from the group consisting of dipeptides andtripeptides; or is

or is

where aa is a natural or non-natural amino acid residue; or is

where the

ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon atoms.

In certain embodiments the cleavable linker is derived from a linkerprecursor selected from valine-alanine, valine-glutamic acid,alanine-phenylalanine; phenylalanine-lysine; phenylalanine-homolysine;and glycine-glycine-glycine (Gly-Gly-Gly); or is

or is

where aa is a natural or non-natural amino acid residue; or is

where the

ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon atoms.

In certain embodiments the cleavable linker is

Hydrophilic Groups

Hydrophilic groups facilitate increasing the hydrophilicity of thecompounds described herein. It is believed that increased hydrophilicityallows for greater solubility in aqueous solutions, such as aqueoussolutions found in biological systems. Hydrophilic groups can alsofunction as spacer groups or substituents, which are described infurther detail herein.

In certain embodiments, the hydrophilic group is designated HP and HP¹herein. Useful hydrophilic groups include those described herein. Incertain embodiments, the HP hydrophilic group is a divalentpoly(ethylene glycol). In certain embodiments, the HP hydrophilic groupis a divalent poly(ethylene glycol) according to the formula:

wherein m is an integer from 1 to 12, optionally 1 to 4, optionally 2 to4. In certain embodiments, the HP¹ hydrophilic group is a divalenthydrophilic group or a

where R^(SG) is a monovalent hydrophilic group. In certain embodiments,R^(SG) is a monovalent poly(ethylene glycol). In certain embodiments,R^(SG) is a monovalent poly(ethylene glycol) according to the formula:

wherein R is —H or —CH₃ and m is an integer from 1 to 12, optionally 1to 4, optionally 2 to 4. In certain embodiments, R^(SG) is a monovalentpoly(ethylene glycol) according to the formula:

wherein R is —H or —CH₃ and m is an integer from 1 to 12, optionally 1to 4, optionally 2 to 4; R^(SG) or is —C₁-C₆-alkylene-S(O)₃ ⁻. Incertain embodiments, R^(SG) is a monovalent poly(ethylene glycol)according to the formula:

wherein R is —H or —CH₃ and m is 2 to 4; or is —CH₂CH₂—S(O)₃ ⁻. Incertain embodiments, R^(SG) is a monovalent poly(ethylene glycol)according to the formula:

or R^(SG) is —CH₂CH₂—S(O)₃ ⁻. In certain embodiments, R^(SG) is—C₁-C₆-alkylene-S(O)₃ ⁻. In certain embodiments, R^(SG) is —CH₂CH₂—S(O)₃³¹.

Spacer Groups

Spacer groups facilitate spacing of the conjugating group from the othergroups of the compounds described herein. This spacing can lead to moreefficient conjugation of the compounds described herein to a secondcompound. The spacer group can also stabilize the conjugating group.

In certain embodiments, the spacer group is designated SG herein. Usefulspacer groups include those described herein. In certain embodiments,the spacer group is:

In certain embodiments, SG, W⁴, and the HP or HP¹ group combine to forma divalent poly(ethylene glycol) according to the formula:

wherein m is an integer from 1 to 12, optionally 1 to 4, optionally 2 to4.

Conjugating Groups and Residues Thereof

Conjugating groups facilitate conjugation of the compounds describedherein to a second compound, such as a targeting moiety. In certainembodiments, the conjugating group is designated R herein. Conjugatinggroups can react via any suitable reaction mechanism known to those ofskill in the art. In certain embodiments, a conjugating group reactsthrough a [3+2] alkyne-azide cycloaddition reaction, inverse-electrondemand Diels-Alder ligation reaction, thiol-electrophile reaction, orcarbonyl-oxyamine reaction, as described in detail herein. In certainembodiments, the conjugating group comprises an alkyne, strained alkene,tetrazine, thiol, para-acetyl-phenylalanine residue, oxyamine,maleimide, or azide. In certain embodiments, the conjugating group is:

—N₃, or —SH; wherein R²⁰¹ is lower alkyl. In an embodiment, R²⁰¹ ismethyl, ethyl, or propyl. In an embodiment, R²⁰¹ is methyl.

After conjugation, a divalent residue of the conjugating group is formedand is bonded to the residue of a second compound. The structure of thedivalent residue is determined by the type of conjugation reactionemployed to form the conjugate.

In certain embodiments when a conjugate is formed through a [3+2]alkyne-azide cycloaddition reaction, the divalent residue of theconjugating group comprises a triazole ring or fused cyclic groupcomprising a triazole ring. In certain embodiment when a conjugate isformed through a [3+2] alkyne-azide cycloaddition reaction, the divalentresidue of the conjugating group is:

In certain embodiments when a conjugate is formed through a tetrazineinverse electron demand Diels-Alder ligation reaction, the divalentresidue of the conjugating group comprises a fused bicyclic ring havingat least two adjacent nitrogen atoms in the ring. In certain embodimentswhen a conjugate is formed through a tetrazine inverse electron demandDiels-Alder ligation reaction, the divalent residue of the conjugatinggroup is:

In certain embodiments when a conjugate is formed through athiol-maleimide reaction, the divalent residue of the conjugating groupcomprises succinimidylene and a sulfur linkage. In certain embodimentswhen a conjugate is formed through a thiol-maleimide reaction, thedivalent residue of the conjugating group is:

In certain embodiments when a conjugate is formed through acarbonyl-oxyamine reaction, the divalent residue of the conjugatinggroup comprises a divalent residue of a non-natural amino acid. Incertain embodiments when a conjugate is formed through acarbonyl-oxyamine reaction, the divalent residue of the conjugatinggroup is:

In certain embodiments when a conjugate is formed through acarbonyl-oxyamine reaction, the divalent residue of the conjugatinggroup comprises an oxime linkage. In certain embodiments when aconjugate is formed through a carbonyl-oxyamine reaction, the divalentresidue of the conjugating group is:

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof; wherein Ar is adivalent five- or six-membered, substituted or unsubstituted, monocyclicaryl or heteroaryl ring. In an embodiment, provided herein is a compoundaccording to any of Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein Ar is a divalent six-membered, substituted orunsubstituted, monocyclic aryl or heteroaryl ring. In an embodiment,provided herein is a compound according to any of FormulasXVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof; wherein Ar is adivalent eight-, nine- or ten-membered, substituted or unsubstituted,fused bicyclic aryl or heteroaryl ring. In an embodiment, providedherein is a compound according to any of Formulas XVIb1000-1002b, I-Ib,or X-XIXb-2, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein Ar is a divalentnine-membered, substituted or unsubstituted, fused bicyclic heteroarylring. In an embodiment, provided herein is a compound according to anyof Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Aris phenylene or indolylene, each of which is unsubstituted orsubstituted. In an embodiment, provided herein is a compound accordingto any of Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein Ar is any of the following:

In an embodiment, provided herein is a compound according to any ofFormulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Lis absent. In an embodiment, provided herein is a compound according toany of Formulas XVIb1000-1002b, I-Ib, or X-XIXb-2, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Lis —CH₂—.

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “EG” is present inthe formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein EG comprises phenylene,carboxylene, amino, or a combination thereof. In an embodiment, providedherein is a compound according to any of Formulas I 1000-1002b andI-XIXb-2 in which the group “EG” is present in the formula, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein EG is:

wherein each R^(EG) is independently selected from the group consistingof hydrogen, alkyl, biphenyl, —CF₃, —NO₂, —CN, fluoro, bromo, chloro,alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylaminoC(O)—. In the second and third structures, those of skillwill recognize that EG is bonded to an RT that is not within thebackbone of formula 1000 or (I) as indicated in the above description offormula 1000 and (I). In some embodiments, each R^(EG) is independentlyselected from the group consisting of hydrogen, alkyl, biphenyl, —CF₃,alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylaminoC(O)—. In further embodiments, each R^(EG) is independentlyselected from the group consisting of hydrogen, —NO₂, —CN, fluoro,bromo, and chloro.

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “RT” is present inthe formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein RT comprises a residue of anatural or non-natural amino acid or a residue of a sugar. In anembodiment, provided herein is a compound according to any of FormulasI1000-1002b and I-XIXb-2 in which the group “RT” is present in theformula, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein RT is:

Those of skill will recognize that the first structure is divalent andcan be bonded within the backbone of formula 1000 or (I), and that thesecond structure is monovalent and can be bonded to EG as depicted informula 1000 and (I) above.

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “HP” is present inthe formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein HP comprises poly(ethyleneglycol). In an embodiment, provided herein is a compound according toany of Formulas 1000-1002b and I-XIXb-2 in which the group “HP” ispresent in the formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein HP is:

wherein m is an integer from 1 to 12.

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “SG” is present inthe formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein SG com comprises C₁-C₁₀alkylene, C₄-C₆ alkylene, —C(O)—, or combination thereof. In anembodiment, provided herein is a compound according to any of Formulas1000-1002b and I-XIXb-2 in which the group “SG” is present in theformula, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein SG is:

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “W¹,” “W²,” “W³,”“W⁴,” and/or “W⁵” is present in the formula, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein W¹,W², W³, W⁴, and W⁵ are each independently a single bond, absent, orcomprise a divalent ketone, divalent ester, divalent ether, divalentamide, divalent amine, alkylene, arylene, sulfide, disulfide, —C(O)—, ora combination thereof. In an embodiment, provided herein is a compoundaccording to any of Formulas 1000-1002b and I-XIXb-2 in which the group“W¹,” “W²,” “W³,” “W⁴,” and/or “W⁵” is present in the formula, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein W¹, W², W³, W⁴, and W⁵ are each independently a singlebond, absent, or comprise —C(O)—, —O—, —C(O)NH—, —C(O)NH-alkyl-,—OC(O)NH—, —SC(O)NH—, —NH—, —NH-alkyl-, —N(CH₃)CH₂CH₂N(CH₃)—, —S—,—S—S—, —OCH₂CH₂O—, or a combination thereof.

In an embodiment, provided herein is a compound according to any ofFormulas 1000-1002b and I-XIXb-2 in which the group “R” is present inthe formula, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein R is a conjugating group or aresidue of a conjugating group. In an embodiment, provided herein is acompound according to any of Formulas 1000-1002b and I-XIXb-2 in whichthe group “R” is present in the formula, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Rcomprises an alkyne, strained alkene, tetrazine, thiol,para-acetyl-phenylalanine residue, oxyamine, maleimide, carbonyl alkylhalide, aryl sulfide, or azide. In an embodiment, provided herein is acompound according to any of Formulas 1000-1002b and I-XIXb-2 in whichthe group “R” is present in the formula, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Ris:

—N₃, or —SH; wherein R²⁰¹ is lower alkyl. In an embodiment, providedherein is a compound according to any of Formulas 1000-1002b andI-XIXb-2 in which the group “R” is present in the formula, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein R is:

and

-   -   R²⁰¹ is methyl, ethyl, or propyl. In an embodiment, provided        herein is a compound according to any of Formulas 1000-1002b and        I-XIXb-2 in which the group “R” is present in the formula, or a        pharmaceutically acceptable salt, solvate, stereoisomer, or        tautomer thereof, wherein R is:

-   -    and    -   R²⁰¹ is methyl.

In an embodiment, provided herein is a compound according to any ofFormulas I-IXb, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein W¹, W², W³, W⁴, W⁵, EG, RT,HP, SG, and R combine to form —H.

In an embodiment, provided herein is a compound according to Formula1000a or Formula 1000b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein X, EG, RT, HP, SG, W¹, W², W³, W⁴, W⁵, R, L,and Ar are as described in the context of Formula 1000 and/or any of theembodiments described herein.

In an embodiment, provided herein is a compound according to Formula Iaor Formula Ib:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, SG, W¹, W², W³, W⁴, W⁵, R, L, andAr are as described in the context of Formula I and/or any of theembodiments described herein.

In an embodiment, provided herein is a compound according to Formula1001a or Formula 1001b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵, R, L,and Ar are as described in the context of Formula 1001 and/or any of theembodiments described herein.

In an embodiment, provided herein is a compound according to Formula1002a or Formula 1002b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵, R, L,and Ar are as described in the context of Formula 1002 and/or any of theembodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas II-IX-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,and R are as described in the context of Formula 1000, I, 1001, and/orany of the embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas IIa-IXa-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,and R are as described in the context of Formula 1000, I, 1001, and/orany of the embodiments described herein.In an embodiment, provided herein is a compound according to any ofFormulas IIb-IXb-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,and R are as described in the context of Formula 1000, I, 1001, and/orany of the embodiments described herein.

In an embodiment, provided herein is a compound according to Formula X:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein L and Ar are as described in the context ofFormula I.

In an embodiment, provided herein is a compound according to Formula Xaor Xb:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein L and Ar are as described in the context ofFormula I.

In an embodiment, provided herein is a compound according to any ofFormulas XI-XVI-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,R, L, and Ar are as described in the context of Formula 1000, I, 1001,and/or any of the embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas XIa-XVIa-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,R, L, and Ar are as described in the context of Formula 1000, I, 1001,and/or any of the embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas XIb-XIVb-1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein EG, RT, HP, RT¹, HP¹, SG, W¹, W², W³, W⁴, W⁵,R, L, and Ar are as described in the context of Formula 1000, I, 1001,and/or any of the embodiments described herein.

In one embodiment, the compound of Formula 1000 or 1001 is that where Xis

HP¹ is

and RT¹ is a release trigger group, or a cleavable linker; or HP¹ isabsent and RT¹ is a cleavable linker; and all other groups are asdefined for Formula 1000, 1001, and/or any embodiments described herein.In one embodiment, the compound of Formula 1000 or 1001 is that where Xis

HP¹ is

and RT¹ is a release trigger group, or a cleavable linker; or HP¹ isabsent and RT¹ is a cleavable linker; EG is

and all other groups are as defined for Formula 1000, 1001, and/or anyembodiments described herein. In one embodiment, the compound of Formula1000 or 1001 is that where X is

HP¹ is

and RT¹ is a release trigger group, or a cleavable linker; or HP¹ isabsent and RT¹ is a cleavable linker; EG is

W¹-W⁵ are absent; and all other groups are as defined for Formula 1000,1001, and/or any embodiments described herein. In certain embodiments, Lis absent.

In one embodiment, provided herein is a compound according to Formula1000:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a substituted or unsubstituted indolylene or substituted        or unsubstituted phenylene ring;    -   L is absent or —CH₂—;

-   -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, —C(O)—, —O—, —C(O)O—, —OC(O)—, —C(O)NH—, —C(O)NH-alkyl-,        —OC(O)NH—, —SC(O)NH—, —NH—, —N(alkyl)-, —N(R)-alkylene-N(R)—        (where each R is independently H or alkyl),        —N(CH₃)CH₂CH₂N(CH₃)—, —CH₂—, —CH₂CH₂—, —CH(CH₃)—, —C(CH₃)₂—,        —CH₂CH₂CH₂—, phenylene, —NHCH₂CH₂C(O)—, —C(O)CH₂CH₂NH—, —S—,        —S—S—, —OCH₂CH₂O—, or the reverse thereof;    -   EG is absent, or EG is selected from

wherein each R^(EG) is independently selected from the group consistingof hydrogen, alkyl, biphenyl, —CF₃, —NO₂, —CN, fluoro, bromo, chloro,alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylamino-C(O)—;

-   -   RT when in the backbone is

-   -    and RT when bonded to an EG group is

-   -    wherein each RT is optional;    -   RT¹ is absent,

-   -    valine-alanine, valine-glutamic acid, alanine-phenylalanine;        phenylalanine-lysine; phenylalanine-homolysine; and        glycine-glycine-glycine (gly-gly-gly),

-   -    where aa is a natural or non-natural amino acid residue, or

-   -    where the

-   -    ring is a 4-7 membered heterocyclic ring comprising 3-6 carbon        atoms;    -   HP is absent or

-   -    wherein m is an integer from 1 to 12;    -   HP¹ is absent or

-   -    where R^(HP) is wherein R^(HP) is

-   -    wherein R is —H or —CH₃ and m is an integer from 1 to 12 or        R^(HP) is -alkylene-S(O)₃ ⁻.    -   SG is absent,

-   -    and    -   R is a terminal conjugating group;    -   or, in the alternative, W¹, W², W³, W⁴, W⁵, EG, RT, HP, SG, and        R combine to form —H.

In some embodiments, the compound is that where X is —HP¹—RT¹-EG-,—HP¹—RT¹- where RT¹ is a release trigger group, —HP¹—RT¹- where RT¹ is acleavable linker, —HP¹—RT¹- where RT¹ is a release trigger group, —RT¹-,—RT-, —RT-EG-, RT¹-EG-, or -EG(RT)-; and all other groups are as definedin any of the Formula and/or embodiments described herein. In someembodiments, the compound is that where X is —HP¹—RT¹-EG-, —HP¹-RT¹-where RT¹ is a release trigger group, —HP¹—RT¹- where RT¹ is a cleavablelinker, —HP¹—RT¹- where RT¹ is a release trigger group, —RT¹-, —RT-,—RT-EG-, RT¹-EG-, or -EG(RT)-; the release trigger group facilitatesseparation of a biologically active portion of a compound or conjugatein conjunction with an eliminator group; and all other groups are asdefined in any of the Formula and/or embodiments described herein. Insome embodiments, the compound is that where X is —HP¹—RT¹-EG-,—HP¹—RT¹- where RT¹ is a release trigger group, —HP¹—RT¹- where RT¹ is acleavable linker, —HP¹—RT¹- where RT¹ is a release trigger group, —RT¹-,—RT-, —RT-EG-, RT¹-EG-, or -EG(RT)-; W¹, W⁴, W⁵, and L are independentlya single bond or absent; the release trigger group facilitatesseparation of a biologically active portion of a compound or conjugatein conjunction with an eliminator group; and all other groups are asdefined in any of the Formula and/or embodiments described herein. Insome embodiments, the compound is that where X is —HP¹—RT¹-EG-,—HP¹-RT¹- where RT¹ is a release trigger group, —HP¹—RT¹- where RT¹ is acleavable linker, —HP¹—RT¹- where RT¹ is a release trigger group, —RT¹-,—RT-, —RT-EG-, RT¹-EG-, or -EG(RT)-; W¹, W⁴, W⁵, and L are independentlya single bond or absent; SG is

the release trigger group facilitates separation of a biologicallyactive portion of a compound or conjugate in conjunction with aneliminator group; and all other groups are as defined in any of theFormula and/or embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas 101-108 or 1-8:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof.

In an embodiment, provided herein is a compound according to any ofFormulas 101a-108a or 1a-8a:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof.

In an embodiment, provided herein is a compound according to any ofFormulas 101b-108b or 1-8b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof.

Conjugates

The compounds described herein (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b, or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof) can be reacted with a second compound (e.g., apolypeptide or antibody) to form a conjugate. The second compound can beany compound known to be useful for conjugation to the compoundsdescribed herein (e.g., a compound according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b, ora pharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof). Useful second compounds include polypeptides and antibodies.

Therefore, in an aspect, provided herein is a conjugate comprising acompound described herein (e.g., a compound according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b, ora pharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof) linked to a second compound.

In an embodiment, the conjugate is according to the following FormulaE1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   X is

-   -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is absent or an eliminator group;    -   each RT is a release trigger group, in the backbone of Formula        1000 or bonded to EG, wherein each RT is optional;    -   RT¹ is a release trigger group, or a cleavable linker, or RT¹ is        absent;    -   HP is a single bond, absent, or a divalent hydrophilic group;    -   HP¹ is a single bond, absent, a divalent hydrophilic group, or

-   -    where R^(HP) is a monovalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is a divalent residue of a terminal conjugating group;    -   or, in the alternative, W¹, W², W³, W⁴, W⁵, EG, RT, HP, SG, and        R combine to form —H.

In an embodiment, the conjugate is according to the following FormulaC1:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   COMP is a residue of a second compound;    -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is an eliminator group;    -   each RT is a release trigger group, and one RT is optional;    -   HP is a single bond, absent, or a divalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is a divalent residue of a terminal conjugating group.

In one embodiment, provided herein is a conjugate according to Formula(F1) or (G1):

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein:

-   -   COMP is a residue of a second compound;    -   Ar is a divalent five- or six-membered, substituted or        unsubstituted, monocyclic aryl or heteroaryl ring or a divalent        eight-, nine- or ten-membered, substituted or unsubstituted,        fused bicyclic aryl or heteroaryl ring;    -   L is absent or —CH₂—;    -   W¹, W², W³, W⁴, and W⁵ are each independently a single bond,        absent, or a divalent attaching group;    -   EG is absent or an eliminator group;    -   RT¹ is a release trigger group or a cleavable linker; RT is a        release trigger group bonded to EG; and wherein RT and RT¹ are        optional;    -   HP¹ is single bond, absent, a divalent hydrophilic group, or

-   -    where R^(SG) is a monovalent hydrophilic group;    -   SG is a single bond, absent, or a divalent spacer group; and    -   R is a divalent residue of a terminal conjugating group.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Aris a divalent five- or six-membered, substituted or unsubstituted,monocyclic aryl or heteroaryl ring. In an embodiment, provided herein isa conjugate according to any of Formulas C1-C17b, E1, F1-F17b, andG1-G17b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein Ar is a divalent six-membered, substitutedor unsubstituted, monocyclic aryl or heteroaryl ring. In an embodiment,provided herein is a conjugate according to any of Formulas C1-C17b, E1,F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein Ar is a divalent eight-,nine- or ten-membered, substituted or unsubstituted, fused bicyclic arylor heteroaryl ring. In an embodiment, provided herein is a conjugateaccording to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein Ar is a divalent eight-, nine-membered, substituted orunsubstituted, fused bicyclic heteroaryl ring. In an embodiment,provided herein is a conjugate according to any of Formulas C1-C17b, E1,F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein Ar is phenylene orindolylene, each of which is unsubstituted or substituted. In anembodiment, provided herein is a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof, wherein Ar is any ofthe following:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Lis absent. In an embodiment, provided herein is a conjugate according toany of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Lis —CH₂—.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein EGcomprises phenylene, carboxylene, amine, or a combination thereof. In anembodiment, provided herein is a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof; wherein EG is:

wherein each R^(EG) is independently selected from the group consistingof hydrogen, alkyl, biphenyl, —CF₃, —NO₂, —CN, fluoro, bromo, chloro,alkoxyl, alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylaminoC(O)—. In the second and third structures, those of skillwill recognize that EG is bonded to an RT that is not within thebackbone of formula (I) as indicated in the above description of formula(I). In some embodiments, each R^(EG) is independently selected from thegroup consisting of hydrogen, alkyl, biphenyl, —CF₃, alkoxyl,alkylamino, dialkylamino, alkyl-C(O)O—, alkylamino-C(O)— anddialkylaminoC(O)—. In further embodiments, each R^(EG) is independentlyselected from the group consisting of hydrogen, —NO₂, —CN, fluoro,bromo, and chloro.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein RTcomprises a residue of a natural or non-natural amino acid or a residueof a sugar. In an embodiment, provided herein is a conjugate accordingto any of Formulas C1-C17b, E1, F1-F17b, and G-G17b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein RT is:

Those of skill will recognize that the first structure is divalent andcan be bonded within the backbone of formula 1000 or (I), and that thesecond structure is monovalent and can be bonded to EG as depicted informula (I) and 1000 above.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein HPcomprises poly(ethylene glycol). In an embodiment, provided herein is aconjugate according to any of Formulas C1-C17b, E1, F1-F17b, and G-G17b,or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein HP is:

wherein m is an integer from 1 to 12.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein SGcomprises C₁-C₁₀ alkylene, C₄-C₆ alkylene, —C(O)—, or combinationthereof. In an embodiment, provided herein is a conjugate according toany of Formulas C1-C17b, E1, F1-F17b, and G-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein SGis:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein W¹,W², W³, W⁴, and W⁵ are each independently a single bond, absent, orcomprise a divalent ketone, divalent ester, divalent ether, divalentamide, divalent amine, alkylene, arylene, sulfide, disulfide, —C(O)—, ora combination thereof. In an embodiment, provided herein is a conjugateaccording to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein W¹, W², W³, W⁴, and W⁵ are each independently a singlebond, absent, or comprise —C(O)—, —O—, —C(O)NH—, —C(O)NH-alkyl-,—OC(O)NH—, —SC(O)NH—, —NH—, —NH-alkyl-, —N(CH₃)CH₂CH₂N(CH₃)—, —S—,—S—S—, —OCH₂CH₂O—, or a combination thereof.

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Rcomprises a triazole ring. In an embodiment, provided herein is aconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein R is a triazole ring or fused cyclic groupcomprising a triazole ring. In an embodiment, provided herein is aconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein R is:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Rcomprises a fused bicyclic ring having at least two adjacent nitrogenatoms in the ring. In an embodiment, provided herein is a conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein R is:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Rcomprises a sulfur linkage. In an embodiment, provided herein is aconjugate according to any of Formulas C-C13b, E1, F1-F13b, and G1-G13b,or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof; wherein R is:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein Rcomprises a divalent residue of a non-natural amino acid. In anembodiment, provided herein is a conjugate according to any of FormulasC1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof; wherein R is:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; whereincomprises an oxime linkage. In an embodiment, provided herein is aconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof; wherein R is:

In an embodiment, provided herein is a conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; wherein Ris:

In an embodiment, provided herein is a compound according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof; whereinCOMP is a residue of any compound known to be useful for conjugation tothe modified Hemiasterlin compounds described herein (e.g., a compoundaccording to any of Formulas 1000-1000b, 1001-1001b, 1002-1002b, andI-XIXb-2, 101-111b, or 1-8b, or a pharmaceutically acceptable salt,solvate, stereoisomer, or tautomer thereof). In an embodiment, providedherein is a compound according to any of Formulas C1-C17b, E1, F1-F17b,and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein COMP is a residue of apolypeptide, antibody, or antibody chain. In an embodiment, providedherein is a compound according to any of Formulas C1-C17b, E1, F1-F17b,and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein COMP is a residue of apolypeptide. In an embodiment, provided herein is a compound accordingto any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof; wherein COMP is a residue of an antibody. In an embodiment,provided herein is a compound according to any of Formulas C1-C17b, E1,F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof; wherein COMP is a residue of anantibody chain.

In an aspect, provided herein is a polypeptide conjugate comprising acompound described herein (e.g., a compound according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b, ora pharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof) linked to a polypeptide, wherein the polypeptide conjugate isaccording to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein COMP is a residue of the polypeptide. In an embodiment,provided herein is a polypeptide conjugate according to any of FormulasC1-C15b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof, wherein: COMP is a residue of the polypeptide; andR comprises a triazole ring or fused cyclic group comprising a triazolering. In an embodiment, provided herein is a polypeptide conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the polypeptide; and R is:

In an embodiment, provided herein is a polypeptide conjugate accordingto any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the polypeptide; and R comprisesa fused bicyclic ring, wherein the fused bicyclic ring has at least twoadjacent nitrogen atoms in the ring. In an embodiment, provided hereinis a polypeptide conjugate according to any of Formulas C1-C13b, E1,F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein: COMP is a residue of thepolypeptide; and R is:

In an embodiment, provided herein is a polypeptide conjugate accordingto any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the polypeptide; and R comprisesa sulfur linkage. In an embodiment, provided herein is a polypeptideconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof, wherein: COMP is a residue of the polypeptide; andR is:

In an embodiment, provided herein is a polypeptide conjugate accordingto any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the polypeptide; and R comprisesa divalent residue of a non-natural amino acid. In an embodiment,provided herein is a polypeptide conjugate according to any of FormulasC1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is aresidue of the polypeptide; and R is:

In an embodiment, provided herein is a polypeptide conjugate accordingto any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the polypeptide; and R comprisesan oxime linkage. In an embodiment, provided herein is a polypeptideconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof, wherein: COMP is a residue of the polypeptide; andR is:

In an aspect, provided herein is an antibody conjugate comprising acompound described herein (e.g., a compound according to any of Formulas1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or 1-8b, ora pharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof) linked to an antibody according to any of Formulas C1-C17b, E1,F1-F17b, and G1-G17b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein COMP is a residue of theantibody. In an embodiment, provided herein is an antibody conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the antibody; and R comprises atriazole ring or fused cyclic group comprising a triazole ring. In anembodiment, provided herein is an antibody conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R is:

In an embodiment, provided herein is an antibody conjugate according toany of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R comprises a fused bicyclicring, wherein the fused bicyclic ring has at least two adjacent nitrogenatoms in the ring. In an embodiment, provided herein is an antibodyconjugate according to any of Formulas C1-C13b, E1, F1-F13b, andG1-G13b, or a pharmaceutically acceptable salt, solvate, stereoisomer,or tautomer thereof, wherein: COMP is a residue of the antibody; and Ris:

In an embodiment, provided herein is an antibody conjugate according toany of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R comprises a sulfur linkage. Inan embodiment, provided herein is an antibody conjugate according to anyof Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R is:

In an embodiment, provided herein is an antibody conjugate according toany of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R comprises a divalent residue ofa non-natural amino acid. In an embodiment, provided herein is anantibody conjugate according to any of Formulas C1-C13b, E1, F1-F13b,and G1-G13b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein: COMP is a residue of theantibody; and R is:

In an embodiment, provided herein is an antibody conjugate according toany of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R comprises an oxime linkage. Inan embodiment, provided herein is an antibody conjugate according to anyof Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody; and R is:

In an aspect, provided herein is an antibody chain conjugate comprisinga compound described herein (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b, or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof) linked to an antibody chain according to any ofFormulas C1-C17b, E1, F1-F17b, and G1-G17b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, whereinCOMP is a residue of the antibody chain. In an embodiment, providedherein is an antibody chain conjugate according to any of FormulasC1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is aresidue of the antibody chain; and R comprises a triazole ring or fusedcyclic group comprising a triazole ring. In an embodiment, providedherein is an antibody chain conjugate according to any of FormulasC1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceutically acceptablesalt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is aresidue of the antibody chain; and R is:

In an embodiment, provided herein is an antibody chain conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the antibody chain; and Rcomprises a fused bicyclic ring, wherein the fused bicyclic ring has atleast two adjacent nitrogen atoms in the ring. In an embodiment,provided herein is an antibody chain conjugate according to any ofFormulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody chain; and R is:

In an embodiment, provided herein is an antibody chain conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the antibody chain; and Rcomprises a sulfur linkage. In an embodiment, provided herein is anantibody chain conjugate according to any of Formulas C1-C13b, E1,F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein: COMP is a residue of theantibody chain; and R is:

In an embodiment, provided herein is an antibody chain conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the antibody chain; and Rcomprises a divalent residue of a non-natural amino acid. In anembodiment, provided herein is an antibody chain conjugate according toany of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or a pharmaceuticallyacceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:COMP is a residue of the antibody chain; and R is:

In an embodiment, provided herein is an antibody chain conjugateaccording to any of Formulas C1-C13b, E1, F1-F13b, and G1-G13b, or apharmaceutically acceptable salt, solvate, stereoisomer, or tautomerthereof, wherein: COMP is a residue of the antibody chain; and Rcomprises an oxime linkage. In an embodiment, provided herein is anantibody chain conjugate according to any of Formulas C1-C13b, E1,F1-F13b, and G1-G13b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof, wherein: COMP is a residue of theantibody chain; and R is:

In an embodiment, provided herein is a conjugate according to FormulaC1a or Formula C1b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a conjugate according to thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas and/or embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas C2-C9:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are a described in thecontext of any of the Formulas or embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas C2a-C9a:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are a described in thecontext of any of the Formulas or embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas C2b-C9b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are a described in thecontext of any of the Formulas or embodiments described herein.

In an embodiment, provided herein is a compound according to any ofFormulas C10-C13:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an embodiment, provided herein is a compound according to any ofFormulas C10a-C13a:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an embodiment, provided herein is a compound according to any ofFormulas C10b-C13b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an embodiment, provided herein is a compound according to any ofFormula C14-C17:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵, L,and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an embodiment, provided herein is a compound according to any ofFormula C14a-C17a:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵, L,and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an embodiment, provided herein is a compound according to any ofFormula C14b-C17b:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein COMP, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵, L,and Ar are a described in the context of Formulas C1 and I-XVIb.

In an embodiment, provided herein is a compound according to any of thefollowing Formula:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein all other groups are as defined in any of theFormulas or embodiments herein.

In an aspect, provided herein is a method of producing a conjugate(e.g., according to any of Formulas C1-C17b, E1, F1-F17b, and G1-G17b),comprising contacting a compound described herein (e.g., a compoundaccording to any of Formulas I-XVIIIb, 101-111b, or 1-8b) with a secondcompound under conditions suitable for conjugating the compounddescribed herein with the second compound; wherein the second compoundcomprises a modified amino acid comprising an alkyne, strained alkene,tetrazine, thiol, maleimide, carbonyl, oxyamine, or azide. In anembodiment, the second compound is a polypeptide. In an embodiment, thesecond compound is an antibody.

Conjugation Reactions [3+2] Alkyne-Azide Cycloaddition Reaction

Advantageously, the compounds described herein comprising a terminalconjugating alkyne group or an azide group (e.g., a compound accordingto any of Formulas I-IXb, XI-XVIIb, and 101-111b) facilitate selectiveand efficient reactions with a second compound comprising acomplementary azide group or alkyne group. It is believed the azide andalkyne groups react in a 1,3-dipolar cycloaddition reaction to form a1,2,3-triazolylene moiety which links the compound described hereincomprising an alkyne group or an azide group to the second compound.This reaction between an azide and alkyne to form a triazole isgenerally known to those in the art as a Huisgen cycloaddition reactionor a [3+2] alkyne-azide cycloaddition reaction.

The unique reactivity of azide and alkyne functional groups makes themuseful for the selective modification of polypeptides and otherbiological molecules. Organic azides, particularly aliphatic azides, andalkynes are generally stable toward common reactive chemical conditions.In particular, both the azide and the alkyne functional groups are inerttoward the side chains of the 20 common amino acids found innaturally-occurring polypeptides. It is believed that, when brought intoclose proximity, the “spring-loaded” nature of the azide and alkynegroups is revealed and they react selectively and efficiently via a[3+2] alkyne-azide cycloaddition reaction to generate the correspondingtriazole. See, e.g., Chin J., et al., Science 301:964-7 (2003); Wang,Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin, J. W., etal., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the [3+2] alkyne-azide cycloaddition reaction involves aselective cycloaddition reaction [see, e.g., Padwa, A., in COMPREHENSIVEORGANIC SYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), pp. 1069-1109;Huisgen, R. in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A.,1984), pp. 1-176] rather than a nucleophilic substitution, theincorporation of non-naturally encoded amino acids bearing azide andalkyne-containing side chains permits the resultant polypeptides to bemodified selectively at the position of the non-naturally encoded aminoacid. Cycloaddition reactions involving azide or alkyne-containingcompounds can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tomoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew. Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, but not limited to, ascorbate, metallic copper, quinine,hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, and anapplied electric potential.

Inverse Electron Demand Ligation Reaction

Advantageously, the compounds comprising a terminal tetrazine orstrained alkene group provided herein facilitate selective and efficientreactions with a second compound comprising a strained alkene ortetrazine group. It is believed that the tetrazine and strained alkenereact in an inverse-demand Diels-Alder reaction followed by aretro-Diels-Alder reaction which links the compounds comprising aterminal tetrazine or strained alkene group provided herein to thesecond compound. The reaction is believed to be specific, with little tono cross-reactivity with functional groups that occur on biomolecules.The reaction may be carried out under mild conditions, for example atroom temperature and without a catalyst. This reaction between atetrazine and a strained alkene is generally known to those in the artas a tetrazine ligation reaction.

Thiol Reactions

Advantageously, the compounds comprising a terminal thiol group orsuitable electrophilic or disulfide-forming group provided hereinfacilitate selective and efficient reactions with a second compoundcomprising a complementary electrophilic or disulfide-forming group orthiol group. These reactions are believed to be selective with little tono cross-reactivity with functional groups that occur on biomolecules.In another embodiment, the thiol reaction does not include reaction of amaleimide group.

Carbonyl-Oxyamine Reaction

Advantageously, the compounds comprising a terminal carbonyl or oxyaminegroup provided herein facilitate selective and efficient reactions witha second compound comprising an oxyamine or carbonyl group. It isbelieved that the carbonyl and oxyamine react to form an oxime linkage.The reaction is believed to be specific, with little to nocross-reactivity with functional groups that occur on biomolecules.

Other Reactions

Other suitable conjugation reactions are described in the literature.See, for example, Lang, K. and Chin, J. 2014, Bioorthogonal Reactionsfor Labeling Proteins, ACS Chem Biol 9, 16-20; Paterson, D. M. et al.2014, Finding the Right (Bioorthogonal) Chemistry, ACS Chem Biol 9,592-605; King, M. and Wagner, A. 2014, Developments in the Field ofBioorthogonal Bond Forming Reactions—Past and Present Trends,Bioconjugate Chem., 2014, 25 (5), pp 825-839; and Ramil, C. P. and Lin,Q., 2013, Bioorthogonal chemistry: strategies and recent developments,Chem Commun 49, 11007-11022.

Releasing Reactions

Releasing Reactions are reactions that act to release a biologicallyactive portion of a compound or conjugate described herein from thecompound or conjugate in vivo and/or in vitro. In certain embodiments,the released biologically active portion is a compound according to anyof Formulas 1-8b, or a pharmaceutically acceptable salt, solvate,stereoisomer, or tautomer thereof. One example of a releasing reactionis an intramolecular reaction between an eliminator group and a releasetrigger group of a compound or conjugate described herein to release abiologically active portion of a compound or conjugate described herein.The eliminator group may itself devolve into two reactive components, asexemplified in these reactions where X− is a drug having a heteroatom Nor O for linkage. Exemplary Releasing Reactions are depicted in theschemes below:

Compositions

The compounds and conjugates described herein can be formulated intocompositions using methods available in the art and those disclosedherein. Any of the compounds and conjugates described herein can beprovided in an appropriate pharmaceutical composition and beadministered by a suitable route of administration.

In an aspect, provided herein is a pharmaceutical compositioncomprising:

-   -   a compound (e.g., a compound according to any of Formulas        1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or        1-8b) or conjugate (e.g., a conjugate according to any of        Formulas C1-C17b, E1, F1-F17b, and G1-G17b) as described herein;        and    -   a pharmaceutically acceptable excipient, carrier, or diluent.

In certain embodiments, the pharmaceutical compositions provided hereinfurther comprise a pharmaceutically acceptable carrier. The carrier canbe a diluent, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The pharmaceutical composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. The pharmaceutical compositions can take the form of solutions,suspensions, emulsions, tablets, pills, capsules, powders,sustained-release formulations and the like. Oral formulations caninclude standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in E. W. Martin, 1990, Remington's PharmaceuticalSciences, Mack Publishing Co.

In some embodiments, the pharmaceutical composition is provided in aform suitable for administration to a human subject. In someembodiments, the pharmaceutical composition will contain aprophylactically or therapeutically effective amount of the polypeptidetogether with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In some embodiments, the pharmaceutical composition is provided in aform suitable for intravenous administration. Typically, compositionssuitable for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Such compositions, however,may be administered by a route other than intravenous administration.

In particular embodiments, the pharmaceutical composition is suitablefor subcutaneous administration. In particular embodiments, thepharmaceutical composition is suitable for intramuscular administration.

Components of the pharmaceutical composition can be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ample of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

In some embodiments, the pharmaceutical composition is supplied as a drysterilized lyophilized powder that is capable of being reconstituted tothe appropriate concentration for administration to a subject. In someembodiments, polypeptides are supplied as a water free concentrate. Insome embodiments, the polypeptide is supplied as a dry sterilelyophilized powder at a unit dosage of at least 0.5 mg, at least 1 mg,at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, atleast 50 mg, at least 60 mg, or at least 75 mg.

In another embodiment, the pharmaceutical composition is supplied inliquid form. In some embodiments, the pharmaceutical composition isprovided in liquid form and is substantially free of surfactants and/orinorganic salts. In some embodiments, the polypeptide is supplied as inliquid form at a unit dosage of at least 0.1 mg/ml, at least 0.5 mg/ml,at least 1 mg/ml, at least 2.5 mg/ml, at least 3 mg/ml, at least 5mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least25 mg/ml, at least 30 mg/ml, or at least 60 mg/ml.

In some embodiments, the pharmaceutical composition is formulated as asalt form. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In therapeutic use, the practitioner will determine the posology mostappropriate according to a preventive or curative treatment andaccording to the age, weight, stage of the disease and other factorsspecific to the subject to be treated. In certain embodiments, doses arefrom about 1 to about 1000 mg per day for an adult, or from about 5 toabout 250 mg per day or from about 10 to 50 mg per day for an adult. Incertain embodiments, doses are from about 5 to about 400 mg per day or25 to 200 mg per day per adult. In certain embodiments, dose rates offrom about 50 to about 500 mg per day are also contemplated.

Methods of Use for Therapy or Prophylaxis

Certain compounds, conjugates, polypeptides, and antibodies providedherein can be used for the treatment or prevention of any disease orcondition deemed suitable to the practitioner of skill in the art.Generally, a method of treatment or prevention encompasses theadministration of a therapeutically or prophylactically effective amountof a compound, conjugate, polypeptide, antibody, or pharmaceuticalcomposition comprising the same to a subject in need thereof to treat orprevent the disease or condition.

In an aspect, provided herein is a method of inhibiting tubulinpolymerization in a subject in need thereof comprising administering aneffective amount of a compound (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b), conjugate (e.g., a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b), or composition comprising thecompound or conjugate, as described herein, to the subject.

In an aspect, provided herein is a method of treating cell proliferationor cancer in a subject in need thereof comprising administering aneffective amount of a compound (e.g., a compound according to any ofFormulas 1000-1000b, 1001-1001b, 1002-1002b, and I-XIXb-2, 101-111b, or1-8b), conjugate (e.g., a conjugate according to any of FormulasC1-C17b, E1, F1-F17b, and G1-G17b), or composition comprising thecompound or conjugate, as described herein, to the subject.

A therapeutically effective amount of the compound, conjugate,polypeptide, antibody, or pharmaceutical composition comprising the sameis an amount that is effective to reduce the severity, the durationand/or the symptoms of a particular disease or condition. The amount ofthe compound, conjugate, polypeptide, antibody, or pharmaceuticalcomposition comprising the same that will be therapeutically effectivein the prevention, management, treatment and/or amelioration of aparticular disease can be determined by standard clinical techniques.The precise amount of the compound, conjugate, polypeptide, antibody, orpharmaceutical composition comprising the same to be administered withdepend, in part, on the route of administration, the seriousness of theparticular disease or condition, and should be decided according to thejudgment of the practitioner and each subject's circumstances.

In some embodiments, the effective amount of the compound, conjugate,polypeptide, antibody, or pharmaceutical composition comprising the sameprovided herein is between about 0.025 mg/kg and about 1000 mg/kg bodyweight of a human subject. In certain embodiments, the compound,conjugate, polypeptide, antibody, or pharmaceutical compositioncomprising the same is administered to a human subject at an amount ofabout 1000 mg/kg body weight or less, about 950 mg/kg body weight orless, about 900 mg/kg body weight or less, about 850 mg/kg body weightor less, about 800 mg/kg body weight or less, about 750 mg/kg bodyweight or less, about 700 mg/kg body weight or less, about 650 mg/kgbody weight or less, about 600 mg/kg body weight or less, about 550mg/kg body weight or less, about 500 mg/kg body weight or less, about450 mg/kg body weight or less, about 400 mg/kg body weight or less,about 350 mg/kg body weight or less, about 300 mg/kg body weight orless, about 250 mg/kg body weight or less, about 200 mg/kg body weightor less, about 150 mg/kg body weight or less, about 100 mg/kg bodyweight or less, about 95 mg/kg body weight or less, about 90 mg/kg bodyweight or less, about 85 mg/kg body weight or less, about 80 mg/kg bodyweight or less, about 75 mg/kg body weight or less, about 70 mg/kg bodyweight or less, or about 65 mg/kg body weight or less.

In some embodiments, the effective amount of compound, conjugate,polypeptide, antibody, or pharmaceutical composition comprising the sameprovided herein is between about 0.025 mg/kg and about 60 mg/kg bodyweight of a human subject. In some embodiments, the effective amount ofa compound, conjugate, polypeptide, antibody, or pharmaceuticalcomposition comprising the same of the pharmaceutical compositionprovided herein is about 0.025 mg/kg or less, about 0.05 mg/kg or less,about 0.10 mg/kg or less, about 0.20 mg/kg or less, about 0.40 mg/kg orless, about 0.80 mg/kg or less, about 1.0 mg/kg or less, about 1.5 mg/kgor less, about 3 mg/kg or less, about 5 mg/kg or less, about 10 mg/kg orless, about 15 mg/kg or less, about 20 mg/kg or less, about 25 mg/kg orless, about 30 mg/kg or less, about 35 mg/kg or less, about 40 mg/kg orless, about 45 mg/kg or less, about 50 mg/kg or about 60 mg/kg or less.

The pharmaceutical composition of the method can be administered usingany method known to those skilled in the art. For example, thepharmaceutical composition can be administered intramuscularly,intradermally, intraperitoneally, intravenously, subcutaneouslyadministration, or any combination thereof. In some embodiments, thepharmaceutical composition is administered subcutaneously. In someembodiments, the composition is administered intravenously. In someembodiments, the composition is administered intramuscularly.

Cancers which can be treated using a compound, conjugate, polypeptide,antibody, or pharmaceutical composition disclosed herein include cancerswhere Her2 is overexpressed, CD7 is overexpressed, Her2 is notoverexpressed, and CD7 is not overexpressed, In some embodiments, thecancer is small cell lung cancer, non-small cell lung cancer, ovariancancer, platinum-resistant ovarian cancer, ovarian adenocarcinoma,endometrial cancer, breast cancer, breast cancer which overexpressesHer2, triple-negative breast cancer, a lymphoma, large cell lymphoma,diffuse mixed histiocytic and lymphocytic lymphoma, follicular B celllymphoma, colon cancer, colon carcinoma, colon adenocarcinoma,colorectal adenocarcinoma, melanoma, prostate, or multiple myeloma. Incertain embodiments, the cancer is breast cancer, lung cancer, ovariancancer, endometrial cancer, prostate cancer, colon cancer, colorectalcancer, melanoma, prostate cancer, or multiple myeloma.

Assay Methods

Compounds, conjugates, polypeptides, antibodies, and pharmaceuticalcomposition comprising the same described herein can be assayed fortheir expected activity, or for a new activity, according to any assayapparent to those of skill in the art. The compound, conjugate,polypeptide, antibody, or pharmaceutical composition comprising the samecan be assayed for activity in a functional assay or by quantitating theamount of protein present in a non-functional assay, e.g.immunostaining, ELISA, quantitation on Coomasie or silver stained gel,etc., and determining the ratio of biologically active protein to totalprotein.

The amount of protein produced in a translation reaction can be measuredin various fashions. One method relies on the availability of an assaywhich measures the activity of the particular protein being translated.An example of an assay for measuring protein activity is a luciferaseassay system, or chloramphenical acetyl transferase assay system. Theseassays measure the amount of functionally active protein produced fromthe translation reaction. Activity assays will not measure full lengthprotein that is inactive due to improper protein folding or lack ofother post translational modifications necessary for protein activity.

Another method of measuring the amount of protein produced in coupled invitro transcription and translation reactions is to perform thereactions using a known quantity of radiolabeled amino acid such as³⁵S-methionine, ³H-leucine or ¹⁴C-leucine and subsequently measuring theamount of radiolabeled amino acid incorporated into the newly translatedprotein. Incorporation assays will measure the amount of radiolabeledamino acids in all proteins produced in an in vitro translation reactionincluding truncated protein products. The radiolabeled protein may befurther separated on a protein gel, and by autoradiography confirmedthat the product is the proper size and that secondary protein productshave not been produced.

Preparation of Modified Hemiasterlin Compounds

The compounds provided herein can be prepared, isolated or obtained byany method apparent to those of skill in the art. Compounds providedherein can be prepared according to the General Preparation Schemeprovided herein. Reaction conditions, steps and reactants not providedin the General Preparation Scheme would be apparent to, and known by,those skilled in the art in light of the Examples provided herein.

In the General Preparation Scheme R, SG, HP, RT, EG, W¹, W², W³, W⁴, W⁵,L, and Ar are a described in the context of Formulas C1 and 1000-1000b,1001-1001b, 1002-1002b, and I-XIXb-2.

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of Biological Chemistryand/or the Journal of the American Chemical Society.

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCelsius). All methods are conducted at room temperature (“rt” or“r.t.”), unless otherwise noted.

Example 1a Synthesis of Compound 1 (Two Diastereomers)

Preparation of Compound B2

To a mixed solvent of dichloromethane (100 mL) and 2N HCl (78 mL, 156mmol) at −5° C. was added cold bleach (contain 6% NaOCl, 108 mL, 87mmol) in portions. The mixture was stirred at 0° C. (inside temperature)for 5 min. Sodium 2-mercaptobezothioazole (B, 5 g, 26 mmol) was thenadded into the mixture in multi-portions. The mixture stirred at −5 to−10° C. for 20 min. The organic layer (B1, major is BtsCl) was collectedand mixed with L-valinol (3.2 g, 31.2 mmol) and triethyl amine (8.7 mL,121 mmol) in dichloromethane at r.t. The mixture allowed stirring atr.t. for 1 h. Solvent was removed and product was purified by silica gelcolumn (Hexanes:Ethyl acetate=1:1) to give product B2 (3.1 g, 40%, twosteps) as white solid.

LC-MS (ESI): 301 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 8.08 (dd, J=2.1 and 7.2 Hz, 1H), 7.96 (dd,J=1.8 and 6.9 Hz, 1H), 7.58 (m, 2H), 5.46 (br s, 1H), 3.67 (d, J=4.5 Hz,2H), 3.54 (br s, 1H), 3.23 (brs, 1H), 1.93 (m, 1H), 0.97 (d, J=6.9 Hz,6H).

Preparation of Compound B3

To a solution of B2 (3 g, 10 mmol, 1.0 eq) in dimethylformamide (50 mL)was added potassium carbonate (2.77 g, 20 mmol, 2.0 eq) and iodomethane(1.25 mL, 20 mmol, 2.0 eq) at rt. The mixture was heated to 35° C., 4 h.The solvent was removed and the residue was worked up with ethyl acetateand water (3×), dried with Na₂SO₄ and concentrated to give product B3(3.14 g, 100%) as white solid.

LC-MS (ESI): 315 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 8.09 (dd, J=1.6 and 7.5 Hz, 1H), 7.95 (dd,J=1.8 and 6.9 Hz, 1H), 7.58 (m, 2H), 4.25 (br s, 1H), 2.90 (s, 3H), 1.93(m, 1H), 1.02 (dd, J=2.1 and 6.6 Hz, 6H).

Preparation of Compound B4

To a mixed solvent of dichloromethane (50 mL) and DMSO (1.56 mL, 22mmol, 2.2 eq) at −78° C. was added oxalyl chloride (1.05 mL, 12 mmol,1.2 eq) slowly under nitrogen and stirred at this temperature for 30min. B3 (3.14 g, 10 mmol, 1.0 eq) in 20 mL of dichloromethane was thenadded into this reaction mixture at −78° C. under nitrogen. The reactionmixture allowed stirring at −78° C. for 2 h. Triethylamine (7 mL, 50mmol, 5 eq) was then added into the reaction and stirred at −78° C. for30 min. and continued to warm up to 0° C. for another 30 min. Thereaction mixture was poured into an ice-water and extracted with DCM(3×). The organic layer was washed with half saturated ammonium chloride(2×) solution, brine and dried with sodium sulfate. It was concentratedat low temperature (below 30° C.) to give product B4 (3.0 g, 96%) aswhite solid.

¹H NMR (300 MHz, CDCl₃) δ 9.69 (s, 1H), 8.17 (dd, J=1.5 and 8.1 Hz, 1H),7.95 (dd, J=2.1 and 6.9 Hz, 1H), 7.58 (m, 2H), 4.30 (d, J=10.2 Hz, 1H),3.01 (s, 3H), 2.21 (m, 1H), 1.15 (d, J=6.6 Hz, 3H), 0.98 (d, J=6.6 Hz,3H).

Preparation of Compound B5

Product B4 (3 g, 9.58 mmol, 1.0 eq) and[(1-ethoxycaarbonyl)ethylidene]Ph₃P (6.95 g, 19.2 mmol, 2 eq) weredissolved in anhydrous tetrahydrofuran (60 mL) and was heated to reflux,3 h. The reaction was cooled to r.t. and poured into ice water. Productwas extracted with ethyl acetate (3×). The organic layer was washed withbrine, dried with sodium sulfate and then concentrated to give crudeproduct. It was further purified by silica gel column (Hexanes:Ethylacetate=8:2) to give product B5 (2.9 g, 82%).

¹H NMR (300 MHz, CDCl₃) δ 8.09 (dd, J=1.2 and 7.2 Hz, 1H), 7.93 (dd,J=1.8 and 8.1 Hz, 1H), 7.53 (m, 2H), 6.39 (dd, J=1.6 and 10.5 Hz, 1H),4.41 (t, J=10.5 Hz, 1H), 3.87 (q, J=7.2 Hz, 2H), 3.08 (s, 3H), 1.85 (s,3H), 1.02-1.08 (m, 6H), 0.83 (d, J=6.9 Hz, 3H).

Preparation of Compound B6

To a solution of product B5 (2.9 g, 7.31 mmol, 1.0 eq) indimethylformamide (30 mL) was added potassium carbonate (4.04 g, 29.2mmol, 4.0 eq) and thiophenol (2.25 mL, 21.9 mmol, 3.0 eq). The reactionstirred at r.t. for 1 h. It was then worked up with diethyl ether andwater (3×). The ether layer was extracted with 1% HCl, the aqueous waswashed with ether. The aqueous layer was neutralized with sodiumbicarbonate to pH 8 and extracted with dichloromethane (3×). The organiclayer was dried with sodium sulfate and concentrated to give pureproduct B6 (1.2 g, 84%) as yellow oil.

LC-MS (ESI): 200 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 6.48 (dd, J=1.2 and 10.2 Hz, 1H), 4.18 (q,J=7.2 Hz, 1H), 3.06 (q, J=6.3 Hz, 2H), 2.30 (s, 3H), 1.86 (d, J=1.8 Hz,2H), 1.72 (m, 1H), 1.28 (t, J=7.2 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H), 0.87(d, J=6.9 Hz, 3H).

Preparation of Compounds Bts-Leu-Cl and B7

This synthesis is fully described in Vedejs and Kongkittingam, “A TotalSynthesis of (−)-Hemiasterlin Using N-Bts Methodology,” J. Org. Chem.2001, 66(22), 7355-7364. A summary is provided below.

To a solution of Bts-Leu (2.4 g, 7.3 mmol, 1.0 eq) in anhydrousdichloromethane (30 mL) at 0° C. was added thionyl chloride (1.6 mL,21.9 mmol, 3.0 eq) under nitrogen. The reaction mixture was refluxed at42° C. for 2 h. It was concentrated and co-evaporated with toluene togive Bt-Leu-Cl as a crude solid and was used in the next step reactionwithout further purification.

To a solution of product B6 (1.2 g, 6.02 mmol) in a mixed solvent ofdichloromethane and water (1:1, 40 mL) at 0° C. was added a solution ofsodium carbonate (1.28 g, 12.04 mmol, 2.0 eq) and sodium bicarbonate(1.32 g, 15.7 mmol. 3.2 eq) under nitrogen. The fresh made Bts-Leu-Cl(from above) in dichloromethane (10 mL) was added into this reactionwith syringe. The mixture stirred at 0-5° C. for 1 h. Product B7 wasextracted with dichloromethane and water (3×), dried with sodium sulfateand concentrated to give crude product B7, which was purified by silicagel column (Hexanes:ethyl acetate=1:1) to give product B7 (1.8 g, 59%)as white solid. LC-MS (ESI): 510 (M+1).

¹H NMR (300 MHz, CDCl₃) δ 8.11 (dd, J=1.5 Hz, 8.7 Hz, 1H), 7.93 (dd,J=1.2 Hz, 8.7 Hz, 1H), 7.58 (m, 2H), 6.52 (dd, J=1.2 Hz, 9.9 Hz, 1H),6.10 (d, J=8.7 Hz, 1H), 4.85 (t, J=10.2 Hz, 1H), 4.47 (d, J=8.7 Hz, 1H),4.16 (m, 2H), 2.94 (s, 3H), 1.82 (d, J=1.2 Hz, 2H), 1.27 (m, 3H), 0.98(s, 6H), 0.63 (d, J=6.6 Hz, 3H), −0.12 (d, J=6.6 Hz, 3H).

Preparation of Compound B8

To a solution of B7 (200 mg, 0.392 mmol, 1.0 eq) in DMF (2 mL) was addedpotassium carbonate (217 mg, 1.57 mmol, 4.0 eq) and thiophenol (121 μL,1.18 mmol, 3.0 eq) under nitrogen at r.t. The reaction mixture wasstirred at rt. for 4 h and LC-MS showed the reaction completed. Thereaction was worked up with water and ether and 10% hydrochloric acid(as the literature described) and pure B8 (100 mg, 82%) obtained. LC-MS(ESI): 313 (M+1). ¹H NMR (300 MHz, CDCl₃) δ 7.99 (s, 1H), 6.63 (dd,J=1.2 Hz, 9.9 Hz, 1H), 5.15 (t, J=9.9 Hz, 1H), 4.19 (m, 2H), 3.45 (s,1H), 2.86-2.94 (m, 6H), 1.89 (m, 3H), 1.70 (s, bro, 2H), 1.28 (t, J=5.7Hz, 3H), −0.86-1.01 (m, 12H).

Preparation of Compound A1

A mixture of 3-bromobenzaldehyde (25.0 g, 135 mmol, 1.0 eq), N-acetylglycine (15.8 g, 135 mmol, 1.0 eq) and sodium acetate (10.6 g, 135 mmol,1.0 eq) were suspended in acetic anhydride (40 mL) and heated withstirring to reflux under N₂ for 5 hr. The resulting solution solidifiedupon cooling to room temperature and was quenched with ice-cold waterand filtered. The solids were washed twice more with water, air driedfor 4 h, then further dried in vacuo to give compound A1 (31 g, 86%).

Preparation of Compound A2

Oxazolone A1 (31 g, 117 mmol, 1.0 eq) in 1.0 N NaOH (175 mL, 175 mmol,1.5 eq) was stirred at 85° C. until a translucent reddish solution wasobtained. The reaction was cooled down to room temperature and acidifiedto pH 1.0 with 5 N HCl to precipitate a brown solid. Concentrated HCl(30 mL) was added to the flask, and the reaction solution diluted toabout 500 mL. A reflux was maintained for another 5 hr. The solids werecollected by filtration and washed with water twice, and dried underhigh vacuum to deliver the crude material A2 (23 g, 81%) which was usedwithout further purification.

Preparation of Compound A3

Pyruvic acid A2 (23 g, 94.7 mmol, 1.0 eq) was dissolved in THF (100 mL)and cooled to 0° C. Methyl iodide (36 g, 256 mmol, 2.7 eq) followed by 5N NaOH (80 mL) were slowly added, and the reaction brought to refluxovernight. The volatiles were stripped off and the residual aqueoussolution was extracted with ethyl acetate, and acidified with 10% HCl at0° C. to pH 1. The resulting aqueous layer was extracted with ethylacetate (2×). The combined organics were washed with brine, dried oversodium sulfate, and purified by column chromatography (EtOAc/hexanes1:1) to yield pure compound A3 (11 g, 43%).

Preparation of Compound A4

A 2 N solution of methylamine (14.4 mL, 28.8 mmol, 2.0 eq) was addedinto a solution of the keto-acid A3 (11 g, 40.6 mmol, 1.0 eq) in THF(100 mL) at room temperature and stirred for 4 hr. An 8 N solution ofpyridine-borane complex (5 mL, 40.6 mmol, 1.0 eq) was added, and themixture heated to 55° C. for 3 hr. The reaction was quenched withmethanol, concentrated, and diluted with THF (50 mL) to form a whiteprecipitate. The white solid precipitate was filtered and dried onvacuum to give compound A4 (5 g, 61%).

Preparation of Compound A5

To a solution of compound A4 (1.0 g, 3.5 mmol, 1.0 eq) and (Boc)₂O (1.15g, 5.24 mmol, 1.5 eq) in THF and water (1:1, 20 mL) was added sodiumhydroxide (280 mg, 6.99 mmol, 2.0 eq). The mixture was heated at 60° C.for 5 h. The reaction mixture was cooled and concentrated. The residualaqueous solution was acidified with 10% HCl at 0° C. to pH 1, andextracted with ethyl acetate (3×). The combined organic layers weredried over sodium sulfate, and purified with flash column chromatographyto give compound A5 (420 mg, 31%).

Preparation of Compound A6

To compound A5 (1.58 g, 4.07 mmol, 1 eq) in toluene (15 mL) in a sealedtube was added ammonium hydroxide (2.7 mL, 40.7 mmol, 10 eq) and copperpowder (39 mg, 0.61 mmol, 0.15 eq). The tube was heated to 100° C.overnight and concentrated to give a residue, which was diluted withaqueous NaHCO₃ and n-butanol. The aqueous layer was extracted withn-butanol. The organic layers were concentrated, and purified by silicagel column (DCM:MeOH:Et3N=9:1:1) to give compound A6 (680 mg, 52%).

Preparation of Compound A7

To a solution of compound A6 (1.42 g, 3.36 mmol, 1 eq) in THF (10 mL)was added Alloc-OSu (1.34 g, 6.72 mmol, 2 eq) and triethylamine (1.4 mL,10.1 mmol, 3 eq). The mixture was stirred at rt overnight. The solventwas removed and the residue was purified by flash column chromatography(DCM:MeOH=9:1) to give compound A7 (1.01 g, 74%).

Preparation of Compound A8

To a solution of compound A7 (41 mg, 0.1 mmol, 1 eq) in dry DCM (1.5 mL)was added B8 (31 mg, 0.1 mmol, 1 eq) and PyBOP (57.2 mg, 0.11 mmol, 1.1eq). The mixture was cooled down to 0° C., and DIEA (49 μL, 0.3 mmol, 3eq) was added. The reaction was stirred at rt overnight, and dilutedwith DCM and washed with water. The aqueous was further extracted withDCM (2×). The organic layers were combined, and dried over sodiumsulfate, concentrated to dryness to give a crude product. It waspurified by pre-HPLC to give A8 (10 mg, 14%) as a mixture of twodiastereoisomers (60:40).

Preparation of Compound A9

To a solution of compound A8 (150 mg, 0.21 mmol, 1.0 eq) and Pd(PPh₃)₄(12.4 mg, 0.011 mmol, 0.05 eq) in THF (10 mL) was added tri-n-butyl-tinhydride (113 μL, 0.43 mmol, 2.0 eq). The mixture was degassed andbackfilled with nitrogen (3×). The reaction was stirred at rt for 6 h.The solvent was removed, and the crude product was purified by silicagel column (DCM:MeOH=9:1) to give A9 (78 mg, 60%) as a mixture of twoisomers.

To a solution of compound A9 (28 mg, 0.046 mmol, 1 eq) in MeOH (1 mL)was added LiOH (10 mg, 0.23 mmol, 5 eq) in water (0.5 mL). The mixturewas stirred at rt overnight. The product was purified by prep-HPLC togive A12 (23 mg, 85%).

To a solution of A12 (11 mg, 0.0187 mmol, 1 eq) in DCM (1 mL) was added10% TFA in DCM (1 mL). The mixture stirred at rt for 4 h. Solvent wasremoved and the crude product 1 was purified by preparative RP-HPLCtwice to give two isomers 1a (0.8 mg), and 1b (1 mg).

Example 1b Synthesis of Compound 101 (Two Diastereomers)

Linkers synthesized from the aryl amine Compound 1 give rise tocleavable Compound 101 which releases the novel aniline parent compoundsas a diastereomeric pair.

Preparation of Compound A10

To an argon-flushed solution of A9 (27 mg, 0.04 mmol) in 1 mL CH₂Cl₂ wasadded 15% w/v phosgene in toluene (0.6 mL, 0.06 mmol). The reactionmixture was heated to 50° C. in a sealed tube for 4 h, cooled to ambienttemperature, and the volatiles removed in vacuo. To the residue wasadded a vacuum-dried solution of Fmoc-valine-citruline-p-aminobenzylalcohol (26 mg, 0.04 mmol) in 1 mL DMF. The reaction mixture was stirredat 45° C. under argon for 6 h, then at ambient temperature for 24 hr.After removal of all volatiles in vacuo the residue was purified onsilica gel (90:10 CH₂Cl₂:MeOH eluent) to give 10 mg (0.008 mmol) A10 asa white solid.

Preparation of Compound A11

To a solution of A10 in CH₂Cl₂ (1 mL) was added piperidine (0.1 mL) andthe reaction mixture was stirred at ambient temperature for 1 hr. Afterremoval of all volatiles in vacuo, to the residue was addedDBCO-succinyl N-hydroxysuccinimidyl ester (3.6 mg, 0.009 mmol), DMF (1mL), and diisopropylethylamine (0.004 mL, 0.02 mmol). The reactionmixture was stirred at ambient temperature for 24 hr. After removal ofall volatiles in vacuo the residue was purified on silica gel (90:10CH₂Cl₂:MeOH eluent) to give 7 mg (0.005 mmol) A11.

Preparation of Compound 101

Compound A11 (7 mg, 0.005 mmol) was dissolved in 3:1:1 THF:MeOH:H₂O (1mL) and the solution cooled to 0° C. Solid LiOH·H₂O (1.7 mg, 0.4 mmol)was added and the reaction mixture stirred at ambient temperatureovernight. A few microliters of glacial acetic acid were added, thevolatiles removed in vacuo, and the free acid 101 was purified byreverse phase-high performance liquid chromatography (RP-HPLC) using anUltro 120 (7 μm), 150×20 mm ID column (water-acetonitrile (10 mm NH₄OAc)solvent system, gradient mode from 10% ACN to 100% ACN in 50 min, 15ml/min). LC-MS (ESI): 1282.6 (M+1), 1182.4 (M-Boc+1).

The N-protected acid of A11 (5 mg, 0.004) was dissolved in CH₂Cl₂ (1 mL)and the solution was cooled to 0° C. To this was added a 0.2 M solutionof HCO₂H in CH₂Cl₂ (0.039 mL) and the reaction mixture allowed to stirat ambient temperature overnight. After the volatiles were removed invacuo, the free amino acid was purified by reverse phase-highperformance liquid chromatography (RP-HPLC) using Ultro 120 (7 μm),150×20 mm ID column (water-acetonitrile (10 mm NH₄OAc) solvent system,gradient mode from 10% ACN to 100% ACN in 50 min, 15 ml/min) to give 3mg (0.0025 mmol, 65%) compound 101 as white solid.

Example 1c Chiral Synthesis of Compound A9a

Linkers synthesized from the aryl amine Compound 1 give rise tocleavable Compound 101 which releases the novel aniline parent compoundsas a diastereomeric pair.

Preparation of Compound D12

3,3-Dimethylacrylic acid, (97%, 15.8 g, 157.9 mmol), AlCl₃ (22 g, 164.9mmol) and DCM (100 mL) were placed in a one-neck round-bottomed flaskunder an argon atmosphere. Bromobenzene (31 g, 197.4 mmol) was addedproducing vigorous bubbling. Upon completion of the bubbling, thereaction mixture was stirred in an oil bath at 65° C. for 1 h and 30 minand overnight at rt under N₂ atm. Reaction was poured in HCl:H₂O (1:1)200 mL slowly, EtOAc (300 mL) was added and the organic phase wasseparated, organics were washed with brine, dried over Na₂SO₄, andconcentrated. The crude mixture ¹H NMR data showed mixture ofm,p-regioisomers. Crude material was crystallized from hexane to givepure 3-(3-bromophenyl)-3-methylbutanoicacid (meta isomer) (14 g, 54.7mmol, 42%) as brown crystals.

¹H NMR (400 MHz, CDCl₃) δ 7.52-7.50 (m, 1H), 7.37-7.35 (m, 1H),7.35-7.30 (m, 1H), 7.23-7.20 (m, 1H), 2.66 (s, 2H), 1.47 (s, 6H).

Preparation of Compound D13

3-(3-bromophenyl)-3-methylbutanoicacid (Compound D12, 7.7 g, 29.94 mmol)was dissolved in 170 ml of THF and cooled to −20° C. Triethylamine (8.3ml, 59.89 mmol) and trimethylacetyl chloride (3.7 ml, 29.94 mmol) wereadded to the reaction flask producing a white precipitate. The resultingmixture was stirred at −20° C. for 1 h under N₂ atm, after which LiCl(1.27 g, 29.94 mmol) and (4S)-(−)-4-isopropyl-2oxazolidinone (3.87 g,29.94 mmol) were added sequentially and the resulting reaction mixturewas stirred at −20° C. for 2 h and overnight at rt under N₂ atm. Waterwas added and the reaction mixture was extracted with EtOAc (2×100 mL).The combined organic extracts were dried over magnesium sulfate andconcentrated in vacuo. The product was purified by flash columnchromatography (silica gel, hexane:EtOAc, 4:1) affording compound D13 asa clear, colorless oil in 87% yield (9.5 g, 25.79 mmol).

¹H NMR (400 MHz, CDCl₃) δ 7.52-7.50 (m, 1H), 7.36-7.31 (m, 2H),7.21-7.18 (m, 1H), 4.25-4.21 (m, 1H, H−4), 4.17-4.09 (m, 2H), 3.42-3.31(m, 2H, H−10), 2.22-2.10 (m, 1H), 1.50 (s, 3H), 1.49 (s, 3H), 0.86 (d,3H, J=6.80 Hz), 0.77 (d, 3H, J=6.80 Hz)

Preparation of Compound D14

Oxazolidinone compound D13 (8.4 g, 22.8 mmol) was dissolved in THF (100ml) under an argon atmosphere, and cooled to −78° C. Potassiumbis(trimethylsilyl)amide (25.1 ml, 1 M in THF, 25.1 mmol) was added andthe resulting solution was stirred at −78° C. for 1 h and 20 min. Asolution of 2,4,6-triisopropylbenzenesulfonyl azide (9.2 g, 29.64 mmol)in THF (40 ml) at −78° C. was added via cannula and after 5 min, thereaction mixture was treated with glacial acetic acid (6.3 ml, 104.9mmol), warmed to 40° C., and stirred for an additional 10 h at rt. Brine(270 ml) and Water (35 ml) were added to the light yellow mixture andthe aqueous phase was extracted with (2×500 ml) diethyl ether. Thecombined organic extracts were washed with a saturated sodium hydrogencarbonate solution (2×110 ml), dried with magnesium sulfate, andconcentrated in vacuo. The product was purified by column chromatography(3:7 EtOAc-hexanes), affording azide compound D14 as a colorless oil(8.1 g, 19.84 mmol) in 87% yield.

¹H NMR (400 MHz, CDCl₃) δ 7.55-7.52 (m, 1H), 7.41-7.39 (m, 1H),7.23-7.20 (m, 2H), 5.67 (s, 1H), 4.21-4.07 (m, 3H), 3.61 (t, 1H, J=8.3Hz), 2.37-2.25 (m, 1H, H−6), 1.56 (s, 3H), 1.54 (s, 3H), 0.89 (d, 3H,J=6.8 Hz), 0.85 (d, 3H, J=7.2 Hz).

Preparation of Compound D15

SnCl₂ (5.5 g, 29.32 mmol) was dissolved in 1,4-Dioxane:H₂O (2:1) 75 mLand the resulted colorless clear solution was cooled to 0° C., to whichcompound D14 (4 g, 9.77 mmol) dissolved in 20 mL of dioxane was added,and the reaction mixture was stirred at rt overnight. Reaction wascooled back to 0° C., NaHCO₃ (4.1 g, 48.86 mmol) and Boc₂O (6.4 g, 29.31mmol) were added sequentially, and the reaction was stirred 1 day at rtunder N₂ atm. Solvent was removed under reduced pressure, extracted withEtOAc (2×300 mL) and the organics layer was washed with brine, driedover Na₂SO₄, concentrated, and purified by column chromatography (3:7EtOAc-hexanes), affording compound D15 as a colorless oil (4.1 g, 8.48mmol) in 87.2% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.45 (bs, 1H), 7.35-7.31(m, 1H), 7.32-729 (m, 1H), 6.17 (d, 1H, J=9.6 Hz), 5.15 (bs, 1H, NH),3.89-3.80 (m, 2H), 3.52 (t, 1H, J=8.3 Hz), 2.33-2.21 (m, 1H), 1.41 (s,3H), 1.39 (s, 9H), 1.38 (s, 3H), 0.80 (d, 3H, J=7.2 Hz), 0.78 (d, 3H,J=6.8 Hz)

Preparation of Compound D16

Oxazolidinone Compound D15 (4.1 g, 8.48 mmol) was dissolved in a mixtureof 4:1 THF:H₂O (50 mL). The solution was cooled to 0° C. Hydrogenperoxide (2.7 ml, 30% aqueous, 25.44 mmol) and lithium hydroxide (610mg, 25.44 mmol) were then added to the oxazolidinone solution andstirred at room temperature overnight. The excess peroxide was quenchedby the slow addition of sodium hydrogen sulfite and stirring wascontinued for 1 hr. The mixture was diluted with EtOAc (50 mL) and H₂O(100 mL), the aqueous phase was separated and acidified with 1.0 M HClat 0° C., and extracted with ethyl acetate (2×200 ml). The organic layerwas washed with brine, dried over magnesium sulfate, and concentrated invacuo to give clear colorless oil (2.9 g, 7.83 mmol, 95%) pure enough touse in the next step without further purification. ¹H NMR (400 MHz,CDCl₃) δ 7.32-7.31 (m, 1H), 7.17-7.10 (m, 2H), 7.00-6.97 (m, 1H), 4.83(d, 1H, J=8.8 Hz), 4.41 (d, 1H, J=8.8 Hz), 1.45 (s, 6H), 1.38 (s, 9H).

Preparation of Compound A5a

Under an argon atmosphere, sodium hydride (60%, 830 mg, 22.64 mmol), acatalytic amount of tetrabutylammonium iodide, followed by methyl iodide(2.0 ml, 32 mmol) were added to a vigorously stirred solution of acidcompound D16 (1.2 g, 3.23 mmol) in 50 ml dry THF. The resultingsuspension was stirred 1 day at room temperature. The excess sodiumhydride was quenched by cautious addition of ice cold water and themixture was acidified by drop wise addition of 1.0 M HCl to pH 3 at 0°C. The acidic mixture was extracted with ethyl acetate (3×100 mL), thecombined organic layer was washed with brine, dried over magnesiumsulfate, and concentrated in vacuo. Purification of acid Compound A5awas performed by silica gel column chromatography (1:2 EtOAc-hexaneswith 1% acetic acid) resulting in a 77% yield (0.74 g, 1.93 mmol, 60%)of a clear colorless oil as mixture of rotamers. ¹H NMR (400 MHz, CDCl₃)δ 7.50-7.49 (m, 1H), 7.37-7.27 (m, 2H), 7.13-7.07 (m, 1H), 5.15 (s,0.65H, H−2), 4.95 (s, 0.35H, H−2), 2.51 (s, 1H, H−6), 2.27 (s, 2H, H-6),1.57 (s, 3H), 1.53-1.38 (m, 12H).

Preparation of Compound A6a

Compound A5a (600 mg, 1.56 mmol, 1 eq) in toluene (10 mL) in a sealedtube was added ammonium hydroxide (3 mL, 15.6 mmol, 10 eq) and copperpowder (20 mg, 0.23 mmol, 0.15 eq). The tube was heated to 100° C.overnight and was cooled to rt, sealed tube cap was carefully released,concentrated to give a residue, which was diluted with aqueous NaHCO₃and n-butanol. The aqueous layer was extracted with n-butanol. Theorganic layers were concentrated, and purified by silica gel column(DCM:MeOH:Et₃N=9:1:1) to give compound A6a (300 mg, 0.930 mmol, 60%).LC-MS (ESI): 323.4 (M+1), 223.5 (M-Boc+1).

Preparation of Compound A7A

To a solution of compound A6a (300 mg, 0.930 mmol, 1 eq) in THF (7 mL)was added Alloc-OSu (199.1 mg, 1.86 mmol, 2 eq) and triethylamine (0.51mL, 3.72 mmol, 4 eq). The mixture was stirred overnight at roomtemperature under N₂ atm. The solvent was removed and the residue waspurified by flash column chromatography (DCM:MeOH=9:1) to give compoundA7A (284 mg, 0.70 mmol, 75%). LC-MS (ESI): 407.4 (M+1), 307.6 (M-Boc+1).

Preparation of Compound A8A

To a solution of compound A7A (220 mg, 0.54 mmol, 1 eq) in dry DCM (5mL) was added B8 (202 mg, 0.65 mmol, 1.2 eq). The mixture was cooled to0° C., and DIEA (49 μL, 0.3 mmol, 3 eq) and PyBop (338 mg, 0.65 mmol,1.2 eq) were added sequentially. The reaction was stirred at rtovernight under N₂ atm, and diluted with DCM and washed with water. Theaqueous was further extracted with DCM (2×50 mL). The organic layerswere combined, dried over sodium sulfate, and concentrated to dryness togive a crude product. This crude product was purified by flash columnchromatography (EtOAc:Hexane=1:1) to give compound A8A (200 mg, 0.28mmol, 53%). LC-MS (ESI): 701.4 (M+1), 601.6 (M-Boc+1).

Preparation of Compound A9a

To a solution of compound A8A (170 mg, 0.24 mmol, 1.0 eq) and Pd(PPh₃)₄(28 mg, 0.024 mmol, 0.1 eq) in DCM (10 mL) was added tri-n-butyl-tinhydride (78 μL, 0.29 mmol, 1.2 eq). The reaction mixture was degassedand backfilled with nitrogen. The reaction was stirred for 3-4 h at rtunder N₂ atm. The solvent was removed, and the crude product waspurified by silica gel column (EtOAc:Hexane=1:1) to give A9a (130 mg,87%) as a clear oil. ¹HNMR (400 MHz, CDCl₃) δ 7.11-7.07 (m, 1H),6.97-6.70 (m, 2H), 6.61-6.48 (m, 2H), 6.03-5.97 (m, 1H), 5.12-4.89 (m,1H), 4.63-4.49 (m, 1H), 4.21-4.01 (m, 2H), 2.93 (s, 3H), 2.83 (m, 3H),1.83-1.81 (bs, 4H), 1.42 (s, 12H), 1.27-1.17 (m, 6H), 0.87-0.0.81 (m,6H), 0.78-0.63 (m, 6H), 0.63 (s, 6H). LC-MS (ESI): 617.6 (M+1).

Example 1d Chiral Synthesis of Compound 101a (Single Diastereomer)

Compound 101a is produced from compound A9a according to Scheme 8.

Preparation of Compound A10a

To an argon-flushed solution of A9a (27 mg, 0.04 mmol) in 1 mL CH₂C12 isadded 15% w/v phosgene in toluene (0.6 mL, 0.06 mmol). The reactionmixture is heated to 50° C. in a sealed tube for 4 h, cooled to ambienttemperature, and the volatiles removed in vacuo. To the residue is addeda vacuum-dried solution of Fmoc-valine-citruline-p-aminobenzyl alcohol(26 mg, 0.04 mmol) in 1 mL DMF. The reaction mixture is stirred at 45°C. under argon for 6 h, then at ambient temperature for 24 hr. Afterremoval of all volatiles in vacuo the residue is purified on silica gel(90:10 CH₂Cl₂:MeOH eluent) to give A10a.

Preparation of Compound A11a

To a solution of A10a in CH₂Cl₂ (1 mL) is added piperidine (0.1 mL) andthe reaction mixture is stirred at ambient temperature for 1 hr. Afterremoval of all volatiles in vacuo, to the residue is added DBCO-succinylN-hydroxysuccinimidyl ester (3.6 mg, 0.009 mmol), DMF (1 mL), anddiisopropylethylamine (0.004 mL, 0.02 mmol). The reaction mixture isstirred at ambient temperature for 24 hr. After removal of all volatilesin vacuo the residue is purified on silica gel (90:10 CH₂Cl₂:MeOHeluent) to give Ala.

Preparation of Compound 101a

Compound A11a (7 mg, 0.005 mmol) is dissolved in 3:1:1 THF:MeOH:H₂O (1mL) and the solution cooled to 0° C. Solid LiOH·H₂O (1.7 mg, 0.4 mmol)is added and the reaction mixture stirred at ambient temperatureovernight. A few microliters of glacial acetic acid are added, thevolatiles removed in vacuo, and the free acid 101a is purified byreverse phase-high performance liquid chromatography (RP-HPLC) using anUltro 120 (7 μm), 150×20 mm ID column (water-acetonitrile (10 mm NH₄OAc)solvent system, gradient mode from 10% ACN to 100% ACN in 50 min, 15ml/min).

The N-protected acid of A11a (5 mg, 0.004) is dissolved in CH₂Cl₂ (1 mL)and the solution is cooled to 0° C. To this is added a 0.2 M solution ofHCO₂H in CH₂Cl₂ (0.039 mL) and the reaction mixture is allowed to stirat ambient temperature overnight. After the volatiles are removed invacuo, the free amino acid is purified by reverse phase-high performanceliquid chromatography (RP-HPLC) using Ultro 120 (7 μm), 150×20 mm IDcolumn (water-acetonitrile (10 mm NH₄OAc) solvent system, gradient modefrom 10% ACN to 100% ACN in 50 min, 15 ml/min) to give compound 101a.

Example 1e Synthesis of Compound (110a)

Compound (110a) is prepared according to Scheme 9.

Preparation of Compound A14

Compound A14 was prepared from compound A13 (using procedures similar tothose described for compound A8a) by the method described for compoundA9a. Yield: 379 mg (45%) as a white foam. MS (ESI) m/z 603 (M+H)⁺; ¹HNMR (400 MHz, CDCl₃) δ: 6.96-7.12 (m, 1H), 6.91 (br d, J=7.5 Hz, 1H),6.83 (br s, 1H), 6.66-6.80 (m, 1H), 6.51-6.65 (m, 1H), 6.41-6.50 (m,1H), 5.95 (br d, J=8.8 Hz, 1H), 5.33 (s, 1H), 4.94-5.11 (m, 1H), 4.92(br s, 1H), 4.58 (br d, J=9.5 Hz, 1H), 4.48 (br d, J=8.5 Hz, 1H), 3.72(br s, 1H), 3.67 (br s, 3H), 2.73-2.96 (m, 7H), 1.75-1.98 (m, 5H), 1.72(br s, 1H), 1.49 (br s, 2H), 1.18-1.45 (m, 15H), 0.60-0.86 (m, 17H); ¹³CNMR (400 MHz, CDCl₃) δ: 171.2, 169.9, 168.2, 168.1, 157.1, 148.8, 146.9,139.2, 139.0, 132.5, 132.2, 129.7, 116.6, 113.4, 79.8, 65.4, 55.9, 55.2,52.0, 42.5, 42.5, 34.7, 34.3, 33.9, 31.0, 31.0, 30.2, 30.1, 28.3, 28.3,26.5, 26.3, 26.2, 19.5, 19.4, 18.8, 18.5, 13.9, 13.8.

Preparation of Compound A15

To an argon-flushed, ice-cooled solution of A14 (63 mg, 0.1 mmol) inCH₂Cl₂ (0.8 mL) was added trifluoroacetic acid (0.2 mL, 2.6 mmol). Theice bath was removed, and the reaction was stirred at ambienttemperature for 1 h. The volatiles were removed in vacuo to give A15 asa pale yellow glass that was used directly in the subsequent reaction.MS (ESI) m/z 503 (M+H)+; ¹H NMR (400 MHz, CDCl₃) δ10.36 (br, 3H),7.19-8.14 (m, 4H), 6.57 (br d, J=7.5 Hz, 1H), 5.02-4.45 (m, 3H), 3.68(s, 3H), 2.97 (s, 3H), 2.38-2.48 (m, 3H), 1.74-1.80 s (m, 3H), 1.18-1.38(m, 6H), 0.80-0.95 (m, 17H); ¹³C NMR (400 MHz, CDCl₃) δ: 170.9, 168.2,161.5, 145.3, 139.2, 138.0, 131.5, 126.8, 122.3, 117.2, 86.9, 52.1,40.9, 40.5, 35.3, 34.4, 33.6, 31.4, 29.7, 28.2, 28.3, 27.5, 26.5, 26.4,26.3, 26.2, 19.1, 187, 18.4, 13.8.

Preparation of Compound A16

Compound A16 was prepared from compound A15 by the general methoddescribed for compound A10. Yield: 52 mg (50%) as a white foam. MS (ESI)m/z 1044 (M+H)+; ¹H NMR (400 MHz, CDCl₃) δ 9.10 (br, 1H), 7.84 (brd,J=7.8 Hz, 1H), 7.62 (d, J=7.6 Hz, 2H), 7.42-7.31 (m, 7H), 7.11-7.23 (m,7H), 6.94-6.98 (m, 1H), 6.56 (d, J=8.0 Hz, 1H), 5.94-5.96 (m, 1H),4.85-5.1 (m, 3H), 4.61-4.80 (m, 2H), 4.15 (t, J=7.3 Hz, 1H), 4.25 (t,J=7.0 Hz, 1H), 4.15 (t, J=7.1 Hz, 1H), 4.05 (t, J=7.0 Hz, 1H), 3.67 (s,3H), 3.60 (dJ=3.6 Hz, 1H), 2.89-3.05 (m, 5H), 1.80=2.09 (m, 10H),1.28-1.36 (m, 10H), 0.66-0.99 (m, 17H); ¹³C NMR (400 MHz, CDCl₃) δ:172.4, 172.0, 171.5, 170.9, 168.2, 156.7, 153.9, 147.9, 143.7, 143.6,141.3, 138.9, 132.3, 129.2, 127.7, 127.0, 125.0, 120.67.1, 66.0, 54.7,52.0, 49.6, 47.1, 41.2, 35.6, 35.1, 31.1, 31.0, 29.8, 29.2, 26.7, 21.7,19.5, 19.3, 19.2, 19.0, 18.9, 18.1, 18.0, 13.8.

Preparation of Compound A17

To an argon-flushed solution of A16 (52 mg, 0.05 mmol) in DMF (0.4 mL)was added N,N-diethylamine (0.2 mL, 5 mmol), and the reaction wasstirred at ambient temperature 2 h. The volatiles were removed in vacuo,and the residue purified on silica gel (Biotage Isolera) using agradient of 2-100% methanol in chloroform to yield 23 mg free amine as awhite solid. This was further purified on RP-HPLC (x mm C18 5 □□□ usinga linear gradient of 10 to 90% B in A over 20 minutes (A=10 mM NH₄OAc inwater; B=10 mM NH₄OAc in CH₃CN) and detected at 254 and 280 nM to giveA17 Yield 9.5 mg, 24%). MS (ESI) m/z 822 (M+H)+; ¹H NMR (400 MHz, CDCl₃)δ 9.11 (s, 1H), 7.73 (br, 1H), 7.81 (d, J=9.5 Hz, 1H), 7.48 (d, J=9.0Hz, 2H), 7.01-7.3 (m, 4H), 6.57 (dd, J=1.4, 9.5 Hz, 1H), 5.07 (s, 2H),5.04 (d, J=1.1 Hz, 1H), 4.72 (d, J=9.8 Hz, 1H), 4.6 (t, J=7.0 Hz, 1H),3.6 (s, 3H), 3.63 (s, 1H), 3.24 (br, 1H), 3.05 (s, 1H), 3.01, (d J=6.3Hz, 1H), 2.95 (s, 3H), 2.08 (br, 2H), 2.01 (s, 3H), 1.98 (s, 3H),1.80-1.84 (m, 1H), 1.40 (d, J=6.2 Hz, 4H), 1.31 (s, 6H), 0.92-0.93 (m,13H), 0.79 (d, J=6.6 Hz, 8H), 0.71 (d, J=6.6 Hz, 4H)

Preparation of (110a)

Under an argon atmosphere, compound A17 (9.8 mg, 0.012 mmol) was stirredwith dibenzocyclooctynyladipoyl N-hydroxysuccinimidyl ester (Broadpharm22447, 7.7 mg, 0.018 mmol) in DMF (0.150 mL). To this was added N,N-diisopropylethylamine (0.006 mL, 0.036 mmol) and the reaction wasstirred at ambient temperature for 3 h. The volatiles were removed invacuo, and the residue partially purified on silica gel using a gradientof 2 to 10% methanol in chloroform. The product-containing fractionswere concentrated in vacuo to a residue MS m/z 1137.4. This residue(10.6 mg, 0.009 mmol), under an argon atmosphere, was dissolved in 3:1:1THF:methanol:water (0.5 mL), cooled in an ice-bath, and treated withLiOH·H₂O (2.6 mg, 0.063 mmol). The reaction mixture was allowed toequilibrate to ambient temperature overnight. Volatiles were removed invacuo and the residue purified by RP-HPLC as above to yield, afterlyophilization, (110a), 2.1 mg, 0.002 mmol, as a flocculent white solid.MS (ESI) m/z 1123 (M+H)+; ¹H NMR (400 MHz, DMSO-d₆) δ: 10.02 (br d,J=3.8 Hz, 1H), 9.67 (br s, 1H), 8.25 (br d, J=6.8 Hz, 1H), 7.88 (br d,J=8.8 Hz, 1H), 7.79 (br d, J=8.3 Hz, 1H), 7.54-7.70 (m, 4H), 7.29-7.54(m, 9H), 7.06-7.27 (m, 3H), 6.40-6.50 (m, 1H), 5.10 (s, 2H), 5.05 (br d,J=14.1 Hz, 1H), 4.91 (br t, J=10.2 Hz, 1H), 4.77 (br d, J=9.5 Hz, 1H),4.25-4.49 (m, 2H), 4.06-4.24 (m, 2H), 3.60-2.99 (m, broad waterenvelope), 2.99 (m, 3H), 2.69-2.82 (m, 3H), 2.56-2.49 (m, DMSOenvelope), 2.42 (br s, 1H), 2.12-2.29 (m, 3H), 1.90-2.11 (m, 6H),1.73-1.90 (m, 6H), 1.15-0.76 (m, 18H), 0.72 (br d, J=6.3 Hz, 6H).

Example 1f Synthesis of Compound (111a)

Compound (111a) was prepared according to Scheme 10.

Preparation of Compound A19: ethyl(S,E)-4-((R)-2-((S)-3-(3-aminophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate

An oven-dried 50 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with compound A18 (200 mg,0.32 mmol, 1.0 eq) dry CH₂Cl₂ (3 mL) and the clear solution was cooledto 0° C. with an ice bath, to this 1 mL of Trifluoroacetic acid wasadded. The reaction mixture was allowed to stir 4 h at room temperature.After which LC-MS showed completion of the reaction. The solvent wasremoved under reduced pressure, and the crude material was lyophilizedfor 16 h to give compound A19 (167 mg, 100%) as an off-white solid.LC-MS (ESI): 517.5 (M+1).

Preparation of Compound A20:ethyl(S,E)-4-((S)-2-((R)-3-(3-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate

To an oven-dried 50 mL single-necked round-bottomed flask equipped witha teflon-coated magnetic stir bar is charged with compound A19 (70 mg,0.135 mmol, 1 eq), Fmoc-Valine-Alanine-OH (67 mg, 0.162 mmol, 1.2 eq)and 1 mL of anhydrous N,N-Dimethylformamide. The resulted clear solutionwas cooled to 0° C. with an ice bath, N,N-Diisopropylethylamine (72 μL,0.405 mmol, 3 eq),1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) (62 mg, 0.162 mmol, 1.2 eq) weresequentially added to the reaction. The reaction mixture was allowed tostir at room temperature overnight under N₂ atmosphere. LC-MS showedcompletion of the reaction. The reaction was quenched by the addition ofsaturated NH₄Cl (10 mL) and then extracted with CH₂Cl₂ (2×100 mL). Theorganic layer was washed with saturated brine (50 mL), dried overanhydrous Na₂SO₄, filtered and then concentrated to dryness at reducedpressure. The crude product was purified by preparative reversephase-high performance liquid chromatography using an Ultro 120 (7 μm)C18Q, 150×20 mmID column. Solvent system used Solvent A: watercontaining 10 mm NH₄OAc; Solvent B: acetonitrile containing 10 mmNH₄OAc., Gradient mode from 10% Solvent B to 90% solvent B, over 20minutes, 10 mL/min), pure fractions were collected and lyophilized togive the compound A20 (86 mg, 0.095 mmol, 70%) as a white solid. LC-MS(ESI): 909.5 (M+1).

Preparation of Compound A21

An oven-dried 25 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with compound A20 (80 mg,0.088 mmol), dry CH₂Cl₂ (2 mL), to this clear solution was addedpiperidine (0.5 mL) and the reaction mixture was stirred at ambienttemperature for 1 h under N₂ atm. LC-MS showed completion of thereaction, all volatiles were removed under reduced pressure. The crudefree amine was purified by preparative reverse phase-high performanceliquid chromatography using an Ultro 120 (7 μm) C18Q, 150×20 mmIDcolumn. Solvent system used Solvent A: water containing 10 mm NH₄OAc;Solvent B: acetonitrile containing 10 mm NH₄OAc., Gradient mode from 10%Solvent B to 90% solvent B, over 20 minutes, 10 mL/min), pure fractionswere collected and lyophilized to give the free amine A20a (51 mg, 0.074mmol, 85%) as a white solid. LC-MS (ESI): 688 (M+1).

An oven-dried 25 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with the free amine A20a (51mg, 0.074 mmol). DBCO adipinyl N-hydroxysuccinimidyl ester (41 mg, 0.096mmol), anhydrous N,N-Dimethylformamide (0.5 mL), andN,N-Diisopropylethylamine (40 μL, 0.22 mmol) were sequentially added.The reaction mixture was flushed with Argon and stirred at ambienttemperature for 3 hours under N₂ atm. LC-MS showed completion of thereaction. After removal of all volatiles in vacuo, the residue waspurified by reverse phase-high performance liquid chromatography using aUltro 120 (7 μm) C18Q, 150×20 mm ID column Solvent system used SolventA: water containing 10 mm NH₄OAc; Solvent B: acetonitrile containing 10mm NH₄OAc., Gradient mode from 10% B Solvent to 90% solvent B, over 20minutes, 10 mL/min), pure fractions were collected and lyophilized togive compound A21a (52 mg, 0.052 mmol, 70%) as a white powder. LC-MS(ESI): 1003.8 (M+1).

Preparation of Compound (111a)

An oven-dried 25 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar is charged with compound A21 (50 mg,0.05 mmoles, 1 equiv.), THF:MeOH:H₂O (3:1:1) (1 mL). The clear solutionwas cooled to 0° C. with an ice bath. Solid LiOH·H₂O (16 mg, 0.349 mmol,7 eq) was added and the reaction was allowed to stir at room temperatureunder N₂ atm for 7 h, after which LC-MS showed completion of thereaction, the volatiles were removed in vacuo, and the crude materialwas purified by reverse phase-high performance liquid chromatographyusing Ultro 120 (7 μm) C18Q, 150×20 mmID column, Solvent system usedSolvent A: water containing 10 mm NH₄OAc; Solvent B: acetonitrilecontaining 10 mm NH₄OAc., Gradient elution mode from 10% Solvent B to90% solvent B, over 20 minutes, 10 mL/min), pure fractions werecollected and lyophilized to give compound (111a) (34 mg, 0.034 mmol,70%) as a white powder. LC-MS (ESI): 974.5 (M+1). ¹HNMR (400 MHz,DMSO-d6) δ 9.78-9.63 (bd, 1H), 8.34-8.10 (m, 1H), 7.82-7.68 (2H),7.58-7.35 (m, 8H), 7.34-7.18 (m, 4H), 7.16-7.08 (m, 4H), 6.53-6.52 (m,1H) 4.98-4.93 (m, 1H), 4.85 (m, 1H), 4.71 (brd, 1H), 4.36-4.26 (m,1.5H), 4.09-4.04 (m, 1H), 3.67-3.47 (m, 2H), 3.12 (brd, 1H), 2.91-2.87(m, 3H), 2.13-2.02 (m, 2H), 1.96-1.78 (m, 9H), 1.74-1.61 (m, 5H),1.31-1.03 (m, 16H), 0.90-0.67 (m, 15H).

Example 1g Synthesis of Compound (109a)

Compound (109a) was prepared according to Scheme 11a and 11b.

Preparation of A22

A22 was prepared by a straightforward adaptation of literature methods.(See, for example, Florent et al 1998, J Med Chem 41, 3572; and Jeffreyet al 2006, Biocong Chem 17, 831). LC-MS: T_(R) 9.21 min. m/z 763(M+H)⁺.

Preparation of compound A23:(2S,3R,5S,6S)-2-(2-(3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-4-((((3-((R)-4-(((S)-1-(((S,E)-6-ethoxy-2,5-dimethyl-6-oxohex-4-en-3-yl)(methyl)amino)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-methyl-3-(methylamino)-4-oxobutan-2-yl)phenyl)carbamoyl)oxy)methyl)-6-methylphenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyltriacetate

To an oven-dried 25 mL pressure vessel equipped with a teflon-coatedmagnetic stir bar is charged with compound A19 (25 mg, 0.048 mmol, 1 eq)and 1 mL of anhydrous CH₂Cl₂, to this was added 15% w/v phosgene intoluene (0.7 mL) at rt. The reaction mixture was flushed with Argon andit was stirred in a sealed vessel for 17 h at rt, then concentratedunder reduced pressure to remove volatiles, and dried under high vacuumfor at least 2 hours. To this residue was added compound A22 (44 mg,0.058 mmol, 1.2 eq). This mixture was dissolved in 1 mL of anhydrousN,N-Dimethylformamide. The reaction mixture was stirred at 45° C. for 2h, then at ambient temperature for 12 h. After removal of all volatilesin vacuo the residue was purified by reverse phase-high performanceliquid chromatography using Ultro 120 (7 μm) C18Q, 150×20 mmID column,Solvent system used Solvent A: water containing 10 mm NH₄OAc; Solvent B:acetonitrile containing 10 mm NH₄OAc., Gradient elution mode from 10%Solvent B to 90% solvent B, over 20 minutes, 10 mL/min), pure fractionswere collected and lyophilized to give compound A23 (25 mg, 0.019 mmol,70%) as a white powder. LC-MS (ESI): 1305.8 (M+1).

Preparation of Compound (109a)

An oven-dried 25 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with compound A23 (23 mg,0.018 mmol), dry CH₂Cl₂ (0.5 mL), to this clear solution was addedpiperidine (0.2 mL) and the reaction mixture was stirred at ambienttemperature for 1 h under N₂ atm, all volatiles were removed underreduced pressure. The crude product was dissolved in THF:MeOH:H₂O(3:1:1) (1 mL). The clear solution was cooled to 0° C. with an ice bath.Solid LiOH·H₂O (8 mg, 0.176 mmol, 10 eq) was added and the reaction wasallowed to stir at room temperature under N₂ atm for 7 h, after whichLC-MS showed completion of the reaction, the volatiles were removed invacuo, and the crude material was purified by reverse phase-highperformance liquid chromatography using Ultro 120 (7 μm) C18Q, 150×20mmID column, Solvent system used Solvent A: water containing 10 mmNH₄OAc; Solvent B: acetonitrile containing 10 mm NH₄OAc., Gradientelution mode from 10% Solvent B to 90% solvent B, over 20 minutes, 10mL/min), pure fractions were collected and lyophilized to give compoundA23a (8 mg, 0.009 mmol, 50%) as a white powder. LC-MS (ESI): 915.7(M+H), 897.7 (M−H₂O+H)

An oven-dried 10 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with the free amine A23a (8mg, 0.009 mmol). DBCO adipinyl N-hydroxysuccinimidyl ester (5 mg, 0.011mmol), anhydrous N,N-Dimethylformamide (0.3 mL), andN,N-Diisopropylethylamine (10 μL, 0.033 mmol) were sequentially added.The reaction mixture was flushed with Argon and stirred at ambienttemperature for 3 hours under N₂ atm. LC-MS showed completion of thereaction. After removal of all volatiles in vacuo, the residue waspurified by reverse phase-high performance liquid chromatography using aUltro 120 (7 μm) C18Q, 150×20 mmID column Solvent system used Solvent A:water containing 10 mm NH₄OAc; Solvent B: acetonitrile containing 10 mmNH₄OAc., Gradient mode from 10% B Solvent to 90% solvent B, over 20minutes, 10 mL/min), pure fractions were collected and lyophilized togive compound (109a) (5 mg, 0.004 mmol, 50%) as a white powder. LC-MS(ESI): 1230.8 (M+H), 1212.8 (M−H₂O+H). ¹HNMR (400 MHz, DMSO-d6) δ9.61-9.55 (m, 2H), 7.85 (bs, 2H), 7.80 (bd, 1H), 7.61-6.97 (m, 15H),6.64-6.50 (m, 2H), 6.02 (bs, 1H), 5.48 (bs, 1H), 5.17-4.89 (m, 6H),4.85-4.76 (bt, 1H), 4.71-4.62 (m, 1H), 4.01 (bs, 1H), 3.69-3.66 (m, 3H),3.10 (bs, 2H), 2.89 (bs, 3H), 2.29-2.26 (m, 19H), 2.13-2.00 (m, H),1.96-1.56 (m, 18H), 1.80-1.02 (m, 14H), 0.89 (bs, 10H), 0.70 (d, 3H),0.66 (d, 3H).

Example 1h Synthesis of Compound (111a)

Compound (111a) was prepared according to Schemes 12 and 13.

Preparation of Compound A8a: ethyl(6S,9S,12S,E)-6-(2-(3-(((allyloxy)carbonyl)amino)phenyl)propan-2-yl)-9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate

An oven-dried 100 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar is charged with compound A7a (1.1 g,2.70 mmoles, 1 equiv, prepared internally), dry CH₂Cl₂ (10 mL) andcompound B8 (1.00 g, 3.24 mmoles, 1.2 equiv., prepared internally) indry CH₂Cl₂ (10 mL). The resulted clear solution was cooled to 0° C. withan ice bath, N,N-Diisopropylethylamine (1.5 mL, 8.1 mmoles, 3 equiv.)and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(PyBOP, 1.7 g, 3.24 mmol, 1.2 equiv.) were sequentially added to thecooled solution. The reaction mixture was allowed to stir at roomtemperature overnight under N₂ atmosphere. LC-MS showed completion ofthe reaction. The reaction was quenched by the dropwise addition ofsaturated NH₄Cl (10 mL) and then extracted with CH₂Cl₂ (2×200 mL). Theorganic layer was washed with saturated brine (50 mL), dried overanhydrous Na₂SO₄, filtered and then concentrated to dryness at reducedpressure. The crude product was purified by flash silica columnchromatography on a Teledyne ISCO system (40 g silica flash column,gradient: Hexane to 30% EtOAc/Hexane) to give compound A8a (1.2 g, 1.71mmol, 63%) as a colorless viscous oil which slowly solidifies to give anoff-white solid upon standing. LC-MS (ESI): 701.5 (M+1).

Preparation of Compound A9a: Ethyl(6R,9S,12S,E)-6-(2-(3-aminophenyl)propan-2-yl)-9-(tert-butyl)-12-isopropyl-2,2,5,11,14-pentamethyl-4,7,10-trioxo-3-oxa-5,8,11-triazapentadec-13-en-15-oate

An oven-dried 100 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar is charged with compound A8a (1.2 g,1.71 mmols, 1.0 equiv, prepared internally), dry CH₂Cl₂ (10 mL). To thisclear solution, tetrakis(triphenylphosphine)palladium(0) (0.98 g, 0.856mmoles, 0.5 equiv.) and tri-n-butyl-tin hydride (0.55 mL, 2.05 mmoles,1.2 equiv.) were sequentially added at room temperature under N₂atmosphere. Upon completion of addition, the reaction mixture wasflushed with argon and then stirred at room temperature under a N₂atmosphere. LC-MS & TLC (1:1 EtOAc/Hexane) showed the reaction wascomplete in 4 hours. The solvent was removed under reduced pressure andthe resulting crude material was purified by flash column chromatographyon a Teledyne ISCO system (40 g silica flash column, gradient: Hexane to50% EtOAc/Hexane) to give compound A9a (0.95 g, 1.54 mmole, yield: 90%)as a foamy off-white solid. LC-MS (ESI): 617.3 (M+1).

Preparation of Compound A19: ethyl(S,E)-4-((R)-2-((S)-3-(3-aminophenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate

An oven-dried 50 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with compound A9a (200 mg,0.32 mmol, 1.0 eq) dry CH₂Cl₂ (3 mL) and the clear solution was cooledto 0° C. with an ice bath, to this 1 mL of Trifluoroacetic acid wasadded. The reaction mixture was allowed to stir 4 h at room temperature.After which LC-MS showed completion of the reaction. The solvent wasremoved under reduced pressure, and the crude material was lyophilizedfor 16 h to give compound A19 (167 mg, 100%) as an off-white solid.LC-MS (ESI): 517.5 (M+1).

Preparation of compound A18: ethyl(S,E)-4-((S)-2-((R)-3-(3-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)phenyl)-3-methyl-2-(methylamino)butanamido)-N,3,3-trimethylbutanamido)-2,5-dimethylhex-2-enoate

To an oven-dried 50 mL pressure vessel equipped with a teflon-coatedmagnetic stir bar is charged with compound A19 (167 mg, 0.323 mmol, 1eq) (**Note: compound A19 was dried under lyophilizer pump for 15 hprior to use) and 4 mL of anhydrous CH₂Cl₂ to this was added 15% w/vphosgene in toluene (5 mL) at rt. The reaction mixture was flushed withArgon and it was stirred in a sealed vessel for 17 h at rt, thenconcentrated under reduced pressure to remove volatiles, and dried underhigh vacuum for at least 2 hours. To this residue was added Fmocvaline-citruline-p-aminobenzyl alcohol (253 mg, 0.42 mmol, 1.3 eq). Thismixture was dissolved in 3 mL of anhydrous N,N-Dimethylformamide. Thereaction mixture was stirred at 45° C. for 2 h, then at ambienttemperature for 12 h. After removal of all volatiles in vacuo theresidue was purified by flash column chromatography on a Teledyne ISCO(24 g silica flash column, gradient: CH₂Cl₂ to 12% MeOH/CH₂Cl₂) to givecompound A18 (247 mg, 0.215 mmol, 67%) as an off-white solid. LC-MS(ESI): 1144.7 (M+1).

Preparation of Compound A25

An oven-dried 50 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with compound A18 (240 mg,0.209 mmol), dry CH₂Cl₂ (5 mL), to this clear solution was addedpiperidine (1.5 mL) and the reaction mixture was stirred at ambienttemperature for 1 h under N₂ atm. LC-MS showed completion of thereaction, all volatiles were removed under reduced pressure. The crudefree amine was purified by preparative reverse phase-high performanceliquid chromatography using an Ultro 120 (7 μm) C18Q, 150×20 mmIDcolumn. Solvent system used Solvent A: water containing 10 mm NH₄OAc;Solvent B: acetonitrile containing 10 mm NH₄OAc., Gradient mode from 10%Solvent B to 90% solvent B, over 20 minutes, 10 mL/min), pure fractionswere collected and lyophilized to give the free amine A24 (164 mg, 0.177mmol, 85%) as a white solid.

An oven-dried 50 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar was charged with the free amine A24 (164mg, 0.177 mmol). DBCO succinyl N-hydroxysuccinimidyl ester (126 mg,0.313 mmol), anhydrous N,N-Dimethylformamide (3 mL), andN,N-Diisopropylethylamine (110 μL, 0.627 mmol) were sequentially added.The reaction mixture was flushed with Argon and stirred at ambienttemperature for 3 hours under N₂ atm. LC-MS showed completion of thereaction. After removal of all volatiles in vacuo, the residue waspurified by reverse phase-high performance liquid chromatography using aUltro 120 (7 μm) C18Q, 150×20 mmID column Solvent system used Solvent A:water containing 10 mm NH₄OAc; Solvent B: acetonitrile containing 10 mmNH₄OAc., Gradient mode from 10% B Solvent to 90% solvent B, over 20minutes, 10 mL/min), pure fractions were collected and lyophilized togive compound A25 (177 mg, 0.146 mmol, 70%) as a white powder. LC-MS(ESI): 1209.6 (M+1).

An oven-dried 25 mL single-necked round-bottomed flask equipped with ateflon-coated magnetic stir bar is charged with compound A25 (177 mg,0.146 mmoles, 1 equiv.), THF:MeOH:H₂O (3:1:1) (5 mL). The clear solutionwas cooled to 0° C. with an ice bath. Solid LiOH·H₂O (46 mg, 1.022 mmol,7 eq) was added and the reaction was allowed to stir at room temperatureunder N₂ atm for 7 h, after which LC-MS showed completion of thereaction, the volatiles were removed in vacuo, and the crude materialwas purified by reverse phase-high performance liquid chromatographyusing Ultro 120 (7 μm) C18Q, 150×20 mmID column, Solvent system usedSolvent A: water containing 10 mm NH₄OAc; Solvent B: acetonitrilecontaining 10 mm NH₄OAc., Gradient elution mode from 10% Solvent B to90% solvent B, over 20 minutes, 10 mL/min), pure fractions werecollected and lyophilized to give compound (101a) (138 mg, 0.117 mmol,80%) as a white powder. LC-MS (ESI): 1181.5 (M+1).

Example 1i

Antibodies were expressed in a Xpress CF™ reaction using procedures knowto one of skill in the art. (See, for example, Cai et al. Biotechnol.2015, 31(3), 823; and Zimmerman et al. Bioconjugate Chem. 2014, 25,351.) The cell free extract for this work were created from an OmpTsensitive RF1 attenuated E. coli strain engineered to overexpress E.coli DsbC and FkpA as well as an orthogonal tRNA containing the CUAanti-codon for decoding the Amber Stop Codon. Extract was treated with75 μM iodoacetamide for 45 min at RT (20° C.) and added to a premixcontaining all other components, except for IgG heavy and light chainDNA. The final concentration in the protein synthesis reaction was 30%(v/v) cell extract, 2 mM para-azidomethylphenylalanine (pAMF) (RSP AminoAcids), 5 uM engineered pAMF-specific amino-acyl tRNA synthetase (FRSvariant), 2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium glutamate,130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mMeach of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for Tyrosineand Phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mMspermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 1 μg/mL antiCD74light chain DNA, and 4 μg/mL antiCD74 heavy chain DNA. Site directedmutagenesis was used to introduce an amber stop codon (TAG) into thenucleotide sequence to encode for the pAMF non-natural amino acid atpositions S7 and F404 (light and heavy chains respectively, kabatnumbering). Cell free reactions were initiated by addition of plasmidDNA and incubated at 30° C. for 16 h in 100×10 mm petri dishescontaining 10 mL.

The cell free reactions were clarified by centrifugation at 10,000 rpm'sfor 30 minutes. The clarified supernatant was applied to Protein AMabSelect SuRe (GE Healthcare) with standard wash and low pH elution.Impurities such as aggregates were removed via preparative SEC (SepaxSRT-10C) equilibrated in 50 mM sodium phosphate, 200 mM arginine, pH6.5. Final formulation of the sample was done in Dulbecco's PhosphateBuffered Saline (1×DPBS).

Purified IgGs containing pAMF were conjugated to a cytotoxic testcompound using copper-free click chemistry with strained cyclooctynereagent (SpAAC, strain-promoted alkyne azide cycloadition). In brief,test compounds were dissolved in DMSO to a final concentration of 5 mM.Each compound was added to 1 mg/mL purified protein in PBS at adrug-linker to antibody molar ratio of 12 to 1. The reaction mixture wasincubated at RT (20° C.) for 17 hours. Excess free drug was removed byZeba plate (Thermo Scientific) equilibrated in PBS. DAR analysis wasdone by MALDI-TOF (Bruker AutoFlex Speed). The conjugated protein wasreduced for 10 min at 37° C. with 10 mM TCEP in water and diluted to afinal concentration of 50 μg/mL in 30% acetonitrile, 0.1%trifluoroacetic acid. Samples were combined 1:1 with S-DHB MALDI matrix(50 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid) and 1 μL wasapplied to the MALDI target and dried under vacuum. Each MALDI spectrawas accumulated for 5000 shots at full laser power in linear mode andthe final DAR analysis was calculated by comparing the relative peakintensity for conjugated and unconjugated species.

Example 1j

Conjugates of Compound 1 with trastuzumab were prepared as describedbelow.

Compound 101 or 101a was dissolved in DMSO to a concentration of 5 mM.The solution was added to purified C225 HC C-term antibody in PBS bufferto a final compound concentration of 200 μM and a final antibodyconcentration of 3 mg/mL (20 μM) for a 10:1 molar ratio ofcompound:antibody. The mixture was incubated at ambient temperature (25°C.) for 16 h. The excess compound was removed using zeba plates (ThermoScientific) equilibrated in 1×PBS.

This procedure was used to conjugate compounds 101 and 101a totrastuzumab HC at F404 and to trastuzumab LC at S7.

To make trastuzumab containing a reactive azide group for conjugation,DNA encoding the molecule's, heavy and light chains were cloned into pUGexpression vector. A TAG codon was inserted at the indicated positionsby overlapping PCR. Stop codon TAA was used to terminate translation.

To express protein, cell-free extracts were thawed to room temperatureand incubated with 50 μM iodoacetamide for 30 min. Cell-free reactionswere run at 30 C for up to 16 h containing 30% (v/v)iodoacetamide-treated extract with 8 mM magnesium glutamate, 10 mMammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate,1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids for all18 amino acids except tyrosine and phenylalanine which were added at 0.5mM, 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mMpotassium phosphate, 100 nM T7 RNAP, 2 mM oxidized (GSSG) glutathione, 2mM pAzidoMethylPhenylanine (pAMF), 2.5 μM amber suppressor tRNAsynthetase. The concentrations of heavy chain TAG variant plasmid andwild type light chain plasmid were 7.5 ug/mL and 2.5 ug/mL respectively.

The antibodies containing non natural amino acids were purified byMabSelect and polished by Capto adhere and stored in PBS buffer beforeuse.

The anti-CD74 cell free reactions were clarified by centrifugation at10,000 rpm's for 30 minutes. The clarified supernatant was applied toProtein A MabSelect SuRe (GE Healthcare) with standard wash and low pHelution. Impurities such as aggregates were removed via preparative SEC(Sepax SRT-10C) equilibrated in 50 mM sodium phosphate, 200 mM arginine,pH 6.5. Final formulation of the sample was done in Dulbecco's PhosphateBuffered Saline (1×DPBS).

Example 1k Production of Anti-CD74 Antibodies with Non-Natural AminoAcids

Antibodies were expressed in an Xpress CF™ reaction as describedpreviously with the following modifications. The cell free extract forthis work were created from an OmpT sensitive RF1 attenuated E. colistrain engineered to overexpress E. coli DsbC and FkpA as well as anorthogonal tRNA containing the CUA anti-codon for decoding the AmberStop Codon. Extract was treated with 75 μM iodoacetamide for 45 min atRT (20° C.) and added to a premix containing all other components,except for IgG heavy and light chain DNA. The final concentration in theprotein synthesis reaction was 30% (v/v) cell extract, 2 mMpara-azidomethylphenylalanine (pAMF) (RSP Amino Acids), 5 uM engineeredpAMF-specific amino-acyl tRNA synthetase (FRS variant), 2 mM GSSG, 8 mMmagnesium glutamate, 10 mM ammonium glutamate, 130 mM potassiumglutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP,and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine andPhenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine,15 mM potassium phosphate, 100 nM T7 RNAP, 1 μg/mL antiCD74 light chainDNA, and 4 μg/mL antiCD74 heavy chain DNA. Site directed mutagenesis wasused to introduce an amber stop codon (TAG) into the nucleotide sequenceto encode for the pAMF non-natural amino acid at positions S7 and F404(light and heavy chains respectively, kabat numbering). Cell freereactions were initiated by addition of plasmid DNA and incubated at 30°C. for 16 h in 100×10 mm petri dishes containing 10 mL.

The anti-CD74 cell free reactions were clarified by centrifugation at10,000 rpm's for 30 minutes. The clarified supernatant was applied toProtein A MabSelect SuRe (GE Healthcare) with standard wash and low pHelution. Impurities such as aggregates were removed via preparative SEC(Sepax SRT-10C) equilibrated in 50 mM sodium phosphate, 200 mM arginine,pH 6.5. Final formulation of the sample was done in Dulbecco's PhosphateBuffered Saline (1×DPBS).

Antibodies prepared having non-natural amino acids at positions heavychain residues 404, 241, and 222, according to the EU number scheme, andat light chain residue 7, according to the Kabat or Chothia numberingscheme. One antibody comprised residue (56), above, at position 404, andfour antibodies comprised residue (30), above, at each of positions 404,241, 222 (heavy chain) and 7 (light chain). Each antibody was expressedat a total yield of at least 400 mg/L as shown in FIG. 2A, and intactIgG were detected by SDS-PAGE as shown in FIG. 2B.

Production of Antibody-PEG₄-Maytansine Conjugate

Purified anti-CD74 IgG containing modified amino acid residue 30 (i.e.para-azido-methyl-L-phenylalanine, or pAMF) at EU position 404 in itsheavy chains was obtained according to Example 2. The anti-CD74 IgG wasconjugated to a hemiasterlin, using a strained cyclooctyne reagent toyield Conjugate A.

In brief, DBCO-val-cit-pAB-hemiasterlin according to the following:

was dissolved in DMSO to a final concentration of 5 mM. The compound wasadded to 1 mg/mL purified protein in PBS at a drug to antibody molarratio of 12 to 1. The reaction mixture was incubated at RT (20° C.) for17 hours. Excess free drug was removed by Zeba plate (Thermo Scientific)equilibrated in PBS.

DAR analysis was done by MALDI-TOF (Bruker AutoFlex Speed). Theconjugated protein was reduced for 10 min at 37° C. with 10 mM TCEP inwater and diluted to a final concentration of 50 μg/mL in 30%acetonitrile, 0.1% trifluoroacetic acid. Samples were combined 1:1 withS-DHB MALDI matrix (50 mg/mL in 50% acetonitrile, 0.1% trifluoroaceticacid) and 1 uL was applied to the MALDI target and dried under vacuum.Each MALDI spectra was accumulated for 5000 shots at full laser power inlinear mode and the final DAR analysis was calculated by comparing therelative peak heights for conjugated and unconjugated masses for boththe heavy and light chains.

By peak intensity, MALDI-TOF showed a drug to antibody ratio (DAR) of1.88. Conjugate A, as two regioisomers:

Example 2a Tumor Cell Line Assay

Summary

The two diastereomers of Compound 1, [S,S,S] and [R,S,S] were assayedagainst breast cancer cell lines expressing Her2, CD74 expressing andnon-expressing cell lines, and CD30 expressing and non-expressing celllines. Her2 expressing cell lines included SKBR3, MDA-MB-453, andMDA-MB-468 which are respectively high-, medium-, and low-Her2expressing. CD74 expressing and non-expressing cell lines includedSU-DHL6 and OPM2, which are respectively CD74 expressing andnon-expressing. CD30 expressing and non-expressing cell lines includedL540 and Raji cells, which are respectively CD30 expressing andnon-expressing.

[S,S,S] Compound 1 was found to be 20-fold more potent (average IC₅₀ ca1 nM) against a panel of these tumor cell lines when compared to [R,S,S]Compound 1.

Methods

Cytotoxicity effects of test compounds were evaluated with a cellproliferation assay. Adherent cancer cell lines (SKBR3, MDA-MB435,MDA-MB-468, HCT116, HT29, Skcol, and MDA-MB-453) were obtained from ATCCand maintained in high glucose DMEM/F12 (50/50) medium(Cellgro-Mediatech; Manassas, Va.) supplemented with 10%heat-inactivated fetal bovine serum (Hyclone; Thermo Scientific;Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad, Calif.) and 1×Pencillin/streptomycin (Cellgro-Mediatech; Manassas, Va.). Suspensioncell lines (SU-DHL-6 and OPM-2) were obtained from ATCC and maintainedin high glucose RPMI medium (Cellgro-Mediatech; Manassas, Va.)supplemented with 20% heat-inactivated fetal bovine serum (Hyclone;Thermo Scientific; Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad,Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech; Manassas,Va.).

For adherent cells, a total of 1000 cells in a volume of 40 μL wereseeded in a 96-well half area flat bottom white polystyrene plate theday before the assay. For suspension cells, a total of 20000 cells in avolume of 40 μL were seeded in a 96-well half area flat bottom whitepolystyrene plate on the day of assay.

Testing compounds were formulated at 2× concentration in culture mediumand filtered through MultiScreen HTS 96-Well Filter Plates (Millipore;Billerica, Mass.). Filter sterilized compounds were serial diluted inculture medium and 40 μL of the compounds were added into treatmentwells. For adherent cells, plates were cultured at 37° C. in a CO₂incubator for 96 hours. For suspension cells, the incubation time was 72hours. For cell viability measurements, 80 μL of Cell Titer-Glo® reagent(Promega Corp.; Madison, Wis.) was added into each well, and platesprocessed as per product instructions.

Relative luminescence was measured on an ENVISION® plate reader(Perkin-Elmer; Waltham, Mass.). Relative luminescence readings wereconverted to percent viability using untreated cells as controls. Datawas fitted with non-linear regression analysis, using log(inhibitor) vs.response, variable slope, 4 parameter fit equation using GraphPad Prism(GraphPad v 5.00, Software; San Diego, Calif.). Data was expressed aspercent relative cell viability vs. dose of compounds in nM. Cellkilling IC50 calculated by Prism was used to evaluate the potency ofeach compound on each cell line.

Results

For all the cancer cell lines tested, [S,S,S] Compound 1 (cell killingIC50 ranged from 0.74 nM to 9.18 nM) was found to be 10 to 20-fold morepotent when compared to [R,S,S] Compound 1 (cell killing IC50 rangedfrom 12.21 nM to 91.53 nM).

Results for trastuzumab conjugates are provided in FIG. 1 and Table 1.Results for hemiasterlin derivatives [S,S,S] and [R,S,S] Compound 1, areprovided in FIGS. 2 a-c and Table 2.

TABLE 1 Tumor Cell Line Assay with Trastuzumab Conjugates IC50 TestConjugate (nM) Span (%) Trastuzumab 10.0 66 Trastuzumab F404 [S, S, S]0.2 91 Compound 1, Conjugate Trastuzumab heavy chain F404 0.4 87 racemic[R/S, S, S] Compound 1 conjugate Trastuzumab light chain S7 0.4 86racemic [R/S, S, S] Compound 1 conjugate Trastuzumab heavy chain F4040.1 88 MMAF Conjugate

TABLE 2 Tumor Cell Line Assay with Hemiasterlin Derivatives Cell KillingIC50 (nM) [S, S, S] Origin Cell Line Markers Compound 1 Breast SKBR3Her2 High 1.42 19.97 MDA-MB-453 Her2 Medium 1.80 27.63 MDA-MB-468 Her2Low 0.74 16.71 Colon HCT116 EpCAM High 3.18 62.18 HT29 EpCAM Medium 9.1891.53 Skcol EpCAM Low 3.59 46.88 Melanoma MDA-MB-435 — 2.29 33.74Multiple Myeloma OPM-2 CD74 Negative 2.42 14.35 Lymphoma SU-DHL-6 CD74Positive 1.42 12.21

Example 2b Tumor Cell Line Assay

Cytotoxicity effects of test compounds on target positive and targetnegative cells were measured with a cell proliferation assay. Tumor celllines were obtained from American Type Culture Collection (ATCC) andmaintained in Ham's F-12: high glucose DMEM (50:50) glucose mediumsupplemented with 10% heat-inactivated fetal bovine serum, 1%Penicillin/Streptomcin and 2 mmol/L L-glutamax. Target positive andnegative cells (a total of 625 cells per well) were seeded in a volumeof 25 μL in a 384-well flat bottom white polystyrene plate. The cellswere allowed to adhere overnight at 37° C. in a CO₂ incubator. ADCvariants were formulated at 2× concentration in DMEM/F12 medium andfiltered through MultiScreen HTS 96-Well Filter Plates. Filtersterilized ADCs were serial diluted (1:3) and 25 μL of diluted sampleswere added into each treatment wells. Plates were then cultured at 37°C. in a CO₂ incubator for 120 hrs. For cell viability measurement, 30 μLof Cell Titer-Glo® reagent (Promega Corp) was added into each well, andplates processed as per product instructions. Relative luminescence wasmeasured on an ENVISION® plate reader (Perkin-Elmer; Waltham, Mass.).Relative luminescence readings were converted to % viability usinguntreated cells as controls. Data was fitted with non-linear regressionanalysis, using log(inhibitor) vs. response, variable slope, 4 parameterfit equation using GraphPad Prism (GraphPad v 5.00, Software; San Diego,Calif.). Data was expressed as % relative cell viability vs. dose of ADCin nM.

Results

TABLE 3 Tumor Cell Line Assay with Hemiasterlin Derivatives TargetPositive Cell Target Negative Cell Compound IC50 (nM) Span (%) IC50 (nM)Span (%)  1a 2.2 =90 6.8 =88 101a 222  93 2011 110 110a 26  93 337  90109a 661  92 2440  95 111a 65  92 717  91

TABLE 4 Tumor Cell Line Assay with Antibody Conjugates Target PositiveTarget Negative Cell Cell IC50 Span IC50 Span Conjugate (nM) (%) (nM)(%) Antibody HC-Y180/F404-110a 0.016 74 IA IA Antibody HC-Y180/F404-109a0.039 77 IA IA Antibody HC-Y180/F404-111a IA IA IA IA AntibodyHC-Y180/F404-101a 0.11 73 IA IA IA means not active as tested.

Example 2c Cell Binding and Cell Killing

Conjugate A was evaluated for the ability to bind and kill cellsexpressing CD74 by the methods below. Cell lines tested includedB-lymphoma, multiple myeloma, and leukemia cells. Controls includedunconjugated anti-CD74 antibody.

Cell Binding Assay

Cell lines were maintained in RPMI, high glucose (Cellgro-Mediatech;Manassas, Va.) supplemented with 20% heat-inactivated fetal bovine serum(Hyclone; Thermo Scientific; Waltham, Mass.), 2 mM glutamax (Invitrogen;Carlsbad, Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech;Manassas, Va.). Cells were harvested and re-suspended in FACS buffer(DPBS buffer supplemented with 1% bovine serum albumin). A total of200,000 cells per well were incubated on ice with serial dilutions ofanti-CD74 lead SP7919 without conjugation for 60 minutes. Cells werewashed twice with ice-cold FACS buffer and incubated with 5 ug/ml Alexa647 labeled donkey anti-human IgG antibody (Jackson Immune-Research) onice for another 60 mins. Unstained cells and cells stained withsecondary antibody alone were used as controls. Samples were then washedtwice using FACS buffer and analyzed using a BD FACS Canto system. Meanfluorescence intensities were fitted using non-linear regressionanalysis with one site specific binding equation on GraphPad Prism. Datawas expressed as geometric mean fluorescent intensity vs. antibodyconcentration in nM.

Cell Killing Assay

Cytotoxicity effects of the free drug linkers and conjugates weremeasured with a cell proliferation assay. A total of 12500 cells in avolume of 25 μl were seeded in a 384-well flat bottom white polystyreneplate on the day of assay. Free drug-linkers and conjugates wereformulated at 2× starting concentration (1000 nM for free drug linkersand 100 nM for ADCs) in RPMI medium and filtered through MultiScreen HTS96-Well Filter Plates (Millipore). Filter sterilized conjugated leadswere serial diluted (1:3) under sterile conditions and added intotreatment wells. Plates were cultured at 37° C. in a CO₂ incubator for72 hrs. For cell viability measurement, 30 μl of Cell Titer-Glo® reagent(Promega Corp.) was added into each well, and plates processed as perproduct instructions. Relative luminescence was measured on an ENVISION®plate reader (Perkin-Elmer; Waltham, Mass.). Relative luminescencereadings were converted to % viability using untreated cells ascontrols. Data was fitted with non-linear regression analysis, usinglog(inhibitor) vs. response, variable slope, 4 parameter fit equationusing GraphPad Prism. Data was expressed as % relative cell viabilityvs. dose of free drug-linker or conjugate in nM.

Conjugate A was evaluated for the ability to bind and kill cellsexpressing CD74. Cell lines tested included B-lymphoma, multiplemyeloma, and leukemia cells. Controls included unconjugated anti-CD74antibody. The results are summarized in the following table:

Cell Killing Activity Cell Binding Conjugate A anti-CD74 IC50 DiseaseCell Lines Tested Bmax Kd (nM) (nM) Span (%) B-Lymphoma RPMI-6666 (HL)3879 2.3 0.9 84 SU-DHL-6 (NHL) 1565 2.0 0.3 97 Multiple ARD (MM) 190 2.626.0 74 Myeloma ARP (MM) 7.6 87 RPMI-8226 (MM) 119 3.6 17.0 43 OPM-2(MM) NB NB NK NK Leukemia BDCM (AML) 3059 4.5 1.1 89 SUP-B15 (ALL) 6803.5 2.8 68 JVM-13 (CLL) 447 2.5 0.9 54 K562 (CML) NB NB NK NK

While the claimed subject matter has been described in terms of variousembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the claimed subject matter is limited solely by the scope ofthe following claims, including equivalents thereof.

What is claimed is:
 1. A compound according to Formula 1000:

or a pharmaceutically acceptable salt, solvate, stereoisomer, ortautomer thereof, wherein: Ar is a divalent six-membered, substituted orunsubstituted, monocyclic aryl; divalent five- or six-membered,substituted or unsubstituted, monocyclic heteroaryl; divalent nine- orten-membered, substituted or unsubstituted, monocyclic aryl; or adivalent eight-, nine- or ten-membered, substituted or unsubstituted,fused bicyclic aryl or heteroaryl; L is absent or —CH₂—; X is

R is hydrogen; and W¹, W², W³, W⁴, W⁵, EG, each RT, and HP are allabsent, SG is a single bond.
 2. The compound of claim 1, or apharmaceutically acceptable salt, or tautomer thereof, wherein Ar is adivalent five- or six-membered, substituted or unsubstituted, monocyclicheteroaryl.
 3. The compound of claim 1, or a pharmaceutically acceptablesalt, or tautomer thereof, wherein Ar is a divalent six-membered,substituted or unsubstituted, monocyclic aryl.
 4. The compound of claim1, or a pharmaceutically acceptable salt, or tautomer thereof, whereinAr is a divalent ten-membered, substituted or unsubstituted, fusedbicyclic aryl; or a divalent eight-, nine- or ten-membered, substitutedor unsubstituted, fused bicyclic heteroaryl.
 5. The compound of claim 1,or a pharmaceutically acceptable salt, or tautomer thereof, wherein Aris a divalent nine-membered, substituted or unsubstituted, fusedbicyclic heteroaryl.
 6. The compound of claim 1, or a pharmaceuticallyacceptable salt, or tautomer thereof, wherein Ar is any of thefollowing:


7. The compound of claim 1, or a pharmaceutically acceptable salt, ortautomer thereof, wherein L is absent.
 8. The compound of claim 1, or apharmaceutically acceptable salt, or tautomer thereof, wherein L is—CH₂.
 9. The compound of claim 1, or a pharmaceutically acceptable salt,or tautomer thereof, according to any of the following Formulas:


10. A pharmaceutical composition comprising the compound of claim 1, ora pharmaceutically acceptable salt, or tautomer thereof and apharmaceutically acceptable excipient, carrier, or diluent.
 11. Thepharmaceutical composition of claim 10, wherein the compound is

or a pharmaceutically acceptable salt, or tautomer thereof.
 12. Thecompound of claim 1, wherein the compound is

or a pharmaceutically acceptable salt, or tautomer thereof.